\chapter{Mechanism of HCF-1 cleavage} \label{ch:mechanism}
\thispagestyle{plain}
\vspace{-.5cm}
%
\dsp 
%
\begin{quote}
``In God we trust; all others must bring data.''\newline\\
-- W. Edwards Deming
\end{quote}
%
\noindent A portion of this chapter originally appeared as~\cite{Janetzko:2016er}:\\%\newline\\
``How the glycosyltransferase OGT catalyzes amide bond cleavage''
\textbf{John Janetzko}, Sunia A. Trauger, Michael B. Lazarus and Suzanne Walker
\textit{Nature Chemical Biology}. Volume 12, Number 11: 892--893. 2016.\newline\\
%
Contributions: Except for the following items, I performed all experiments described in this chapter. Michael B. Lazarus expressed, purified, crystallized and solved the structure of the hOGT4.5 (D554N):UDP:glycoCKII ternary complex. Jacob N. Sanders ran computations with my input. Sunia A. Trauger helped me collect and analyze some of the mass spectrometry data. %Jiaoyang Jiang performed the initial \textsuperscript{18}O incorporation experiment.
%
\section*{Abstract}
%
The essential human enzyme OGT, known for modulating the functions of nuclear and cytoplasmic proteins through Ser/Thr glycosylation, was unexpectedly implicated in the proteolytic maturation of the cell cycle regulator HCF-1. In this chapter I will describe my work that showed that HCF-1 cleavage occurs via glycosylation of a glutamate side chain followed by on-enzyme formation of an internal pyroglutamate, which undergoes spontaneous backbone hydrolysis. I will discuss the state of our mechanistic understanding, as well as remaining questions.
%
\section{Introduction}
%
Host cell factor-1 is an essential chromatin-associated protein important in cell cycle control~\cite{Parker:2014eo,Tyagi:2007cf}. It contains six centrally located 26 amino acid proteolytic repeats, each consisting of a threonine-rich region and a region that undergoes cleavage (Figure~\ref{fig:mechanism-hcf-peptide-1})~\cite{Capotosti:2011kd,Wilson:1993wf,Wilson:1995ec}. Proper cell cycle regulation requires HCF-1 cleavage~\cite{Julien:2003in,Mangone:2010cr}. The protease responsible for cleaving HCF-1 went unidentified for many years until studies implicated O-GlcNAc transferase (OGT) in the process~\cite{Capotosti:2011kd,Daou:2011bn}. OGT is an essential nucleocytoplasmic glycosyltransferase best known for its role as a nutrient sensor~\cite{Bond:2015iy,Hardiville:2016jc,Janetzko:2014fy}. The discovery that OGT is involved in HCF-1 processing raised questions about how a glycosyltransferase might promote amide bond hydrolysis.\par
%
\begin{figure}
	\centerline{\includegraphics[width=0.6\columnwidth]{mechanism/hcf-peptide.eps}}
	\caption[Schematic of HCF-1]{HCF-1 contains six similar proteolytic repeats that are cleaved between residues C9 and E10, producing a C-terminal pyroglutamate-containing product, C, that was proposed to form via the intermediacy of a glycosyl ester~\cite{Lazarus:2013bq}.}
	\label{fig:mechanism-hcf-peptide-1}
\end{figure}
%
OGT cleavage of HCF-1 gives a C-terminal product containing an N-terminal pyroglutamate (Figure \ref{fig:mechanism-hcf-peptide-1})~\cite{Lazarus:2013bq}, implying that the cleavage mechanism involves an intermediate in which the glutamate side chain is activated, e.g., through formation of an ester. As UDP-GlcNAc is required for cleavage~\cite{Capotosti:2011kd,Lazarus:2013bq}, but a non-hydrolyzable analog inhibits cleavage~\cite{Lazarus:2013bq}, we proposed that OGT glycosylates the glutamate side chain (Figure \ref{fig:mechanism-hcf-proposed-GP-intermediate}). A mechanism involving glutamate glycosylation would serve to link HCF-1 cleavage to nutrient status through the abundance of UDP-GlcNAc. Other enzymes that use cellular metabolites as co-substrates in amide bond cleavage reactions have been described. For example, sirtuins use NAD\textsuperscript{+} to activate the acetyl-lysine amide bond, thus linking cellular nutrition to protein deacetylation~\cite{Bheda:2016bi,Feldman:2012ez,Verdin:2015hw}. To the best of our knowledge, prior to this work, no NT-sugar glycosyltransferases had been reported to catalyze glycosylation on glutamate, and we did not have direct evidence for this intermediate or for other intermediates that would illuminate the cleavage mechanism.
%
\begin{figure}
	\centerline{\includegraphics[width=0.4\columnwidth]{mechanism/hcf-proposed-GP-intermediate.eps}}
	\caption[Proposed glycosyl glutamate linkage]{Proposed glycosyl glutamate linkage.}
	\label{fig:mechanism-hcf-proposed-GP-intermediate}
\end{figure}
%
\section{Development of an LC-MS-based HCF-1 cleavage assay}
%
In order to identify reaction intermediates, I required an assay with higher resolution than our previous gel-based assay; this would be imperative for the detection of low-abundance molecular species, or those expected to be of (relatively) similar molecular-weight to the starting peptide, and therefore likely indistinguishable by SDS-PAGE. In section~\ref{sec:hcf-peptide-substrate} I showed that a 32 amino acid peptide could serve as a suitable substrate to study HCF-1 cleavage by OGT.\par
%
At the outset of this work, there were two main ideas for how to demonstrate the intermediacy of a glycosyl glutamate linkage. The first, and most compelling, would involve direct observation of such a species. However, given the proposed instability of such an intermediate~\cite{Kotzler:2016br}, a second approach, involving transfer of an \textsuperscript{18}O label from the carboxylate side chain of E10 to the anomeric carbon of GlcNAc would allow us to infer that a covalent bond had formed between them during the cleavage process (Figure~\ref{fig:mechanism-18OGlcNAc}).
%
\begin{figure}[ht]
	\centerline{\includegraphics[width=0.75\columnwidth]{mechanism/18O-GlcNAc-scheme.eps}}
	\caption[Proposed \textsuperscript{18}O transfer experiment]{Proposed \textsuperscript{18}O transfer experiment.~\textsuperscript{18}O labeled atoms are shown in red.}
	\label{fig:mechanism-18OGlcNAc}
\end{figure}
%
Given my intentions of conducting \textsuperscript{18}O experiments, I designed an HCF-1 substrate, E10-32, (See Table~\ref{tab:listofpeptides}) wherein the only carboxylate was that of E10. E13 (of the native rep2 sequence) was substituted with Gln, and the C-terminus was amidated. Further, an N-terminal Tyr was added to allow for absorbance-based standardization of peptide concentration.\par
%
I monitored cleavage E10-32 as a function of time by high-resolution Fourier-transform mass spectrometry (FTMS) (discussed in section~\ref{intro:MS}) and found that under these conditions the reaction was complete within about three hours (Figure \ref{fig:cleavage-reaction}, see also Figure \ref{fig:app-TC1-WT-E10}). Integrating individual extracted ion chromatograms (EICs) shows the disappearance of starting material (fit to a first-order decay, Equation \ref{first-order-decay}), and increases in B and C, which appear to reach a maximal signal around 90 minutes before plateauing (Figure~\ref{fig:cleavage-reaction-plots}).
%
\begin{equation} \label{first-order-decay}
	Y = Ae^{-kX} + b
\end{equation}
%
\begin{figure}[ht]
	\centerline{\includegraphics[width=0.6\columnwidth]{mechanism/cleavage-reaction.eps}}
	\caption[E10-32 undergoes cleavage]{E10-32 undergoes cleavage. A time course for cleavage of E10-32 by wild-type OGT is shown; traces are EICs for the exact masses of species A, B, and C (within $\pm$ 5 ppm). The peptide sequence flanking the cleavage site and the \textit{m/z} of each of the three species are shown under the traces. ``pE'' denotes the pyroglutamate residue. The 32 amino acid cleavage substrate was designed with an N-terminal Y to enable concentration determination, but the sequence shown is numbered starting from the second amino acid to be consistent with the numbering used to describe HCF-1 repeats. The peptide sequence continues after G\textsuperscript{15} as in the repeat in Figure~\ref{fig:mechanism-hcf-peptide-1}; the complete sequence of the cleavage substrate is provided in Table~\ref{tab:listofpeptides}.}
	\label{fig:cleavage-reaction}
\end{figure}
%
\begin{figure}[ht]
	\centerline{\includegraphics[width=0.6\columnwidth]{mechanism/hcf-cleaved-BC-graph.eps}}
	\caption[Plot of EICs for reaction of E10-32 with wild-type OGT]{Plot of EICs for reaction of E10-32 with wild-type OGT. Integration of individual EICs (for data shown in Figure~\ref{fig:app-TC1-WT-E10}), corrected against EIC signal from a blank run.}
	\label{fig:cleavage-reaction-plots}
\end{figure}
%
\section{Detection of HCF-1 cleavage intermediates}
%
Careful examination of the LC-MS traces revealed two low abundance peaks having a \textit{m/z} of 895.9017 (4+), consistent with a glycopeptide [A+GlcNAc] (Figure~\ref{fig:glycopeptides}). One of these peaks formed earlier in the reaction and decayed over time, suggesting it was a glycopeptide intermediate; the other formed more gradually, but increased throughout the cleavage reaction, suggesting it was a glycopeptide byproduct (BP) that prevented backbone cleavage. Glycosylation on T11, the threonine adjacent to the glutamate, or on one of several threonines in the C-terminal region of the peptide that binds to the tetratricopeptide (TPR) domain of OGT, could prevent cleavage by hindering glycosylation at E10.\par
%
\begin{figure}[ht]
	\centerline{\includegraphics[width=0.6\columnwidth]{mechanism/glycopeptides.eps}}
	\caption[Two glycopeptides form during the cleavage reaction]{Two glycopeptides form during the cleavage reaction. LC traces showing EICs for \textit{m/z} 895.9017 $\pm$ 5 ppm, corresponding to [A+GlcNAc]. The y-axis scaling is such that the tallest peak is 100\% (axis not shown). Two peaks having the same exact mass were observed, one of which is a reaction intermediate while the other is a byproduct glycopeptide.}
	\label{fig:glycopeptides}
\end{figure}
%
With the goal of characterizing the glycopeptide intermediate in mind, I sought to eliminate the byproduct glycopeptide that obscured it. Thinking that T11, being adjacent to the reactive glutamate, was a likely off-target glycosylation site I obtained a peptide variant in which T11 was replaced with valine, E10T11V-32. I had previously shown that HCF-3R-EAA (T11V) (see Table~\ref{tab:listhcf3rt11} for sequence) was able to undergo cleavage (see section~\ref{sec:hcf-T11-muts}). A glycopeptide byproduct still formed with this peptide, so we wondered whether it would instead be possible to minimize glycopeptide byproduct formation by using an OGT mutant impaired for Ser/Thr glycosylation, but capable of catalyzing cleavage. I had previously examined a set of OGT active site mutants, and identified one with this behavior (see section~\ref{sec:ogt-muts}).\par
%
We reasoned that a residue that serves the role of a general base for the glycosylation reaction would be dispensable in a scenario where the glycosyl accepter was already deprotonated (e.g., for a carboxylate). Our lab, and others, found that aspartate 554 plays an important role in Ser/Thr glycosylation~\cite{Lazarus:2012gw,Schimpl:2012ka}, and we believed that it was acting (either directly or indirectly) to remove the proton liberated from the acceptor hydroxyl from the active site~\cite{Janetzko:2014fy}. I re-tested the ability of a OGT (D554N) single mutant to catalyze Ser/Thr glycosylation and to effect peptide backbone cleavage using two nearly identical polypeptide substrates, HCF-3R-SAA and HCF-3R-EAA, respectively (see Tables~\ref{tab:listhcf3re10} and \ref{tab:listhcf3rxxx} for sequences, respectively). In one polypeptide, the glutamate in the proteolytic repeat was replaced with serine. Compared to wild-type OGT, OGT (D554N) was dramatically impaired in its ability to glycosylate the serine-containing polypeptide; however, both enzymes were proficient at cleaving the glutamate-containing polypeptide (See Figure~\ref{fig:d554n-cleave-not-glyco}; see previous experiments, Figure~\ref{fig:hcf-ogt-testing}). Over the course of my work, the group of Winship Herr published two papers that made use of OGT (D554H/H558D), a double mutant that seems to show similar behavior to OGT (D554N), albeit with a further reduced ability to catalyze Ser/Thr glycosylation~\cite{Bhuiyan:2015fg,Kapuria:2016gy}.\par
%
\begin{figure}[ht]
	\centerline{\includegraphics[width=0.9\columnwidth]{mechanism/d554n-is-good-1.eps}}
	\caption[OGT (D554N) is impaired for Ser/Thr glycosylation, but not HCF-1 cleavage]{OGT (D554N) is impaired for Ser/Thr glycosylation, but not HCF-1 cleavage. (a) SDS-PAGE analysis detected with Coomassie showed that cleavage of a HCF-3R-EAA construct occurs with both wild-type OGT and the OGT (D554N) mutant. (b) Autoradiograph detecting transfer of \textsuperscript{14}C-GlcNAc to the HCF-3R-SAA polypeptide substrate for wild-type OGT and the D554N mutant. The HCF-3R-AAA peptide was used as a negative control.}
	\label{fig:d554n-cleave-not-glyco}
\end{figure}
%
\begin{figure}[ht]
	\centerline{\includegraphics[width=0.45\columnwidth]{mechanism/enzyme-comparison-100min.eps}}
	\caption[OGT (D554N) does not produce a glycopeptide byproduct]{OGT (D554N) does not produce a glycopeptide byproduct. EICs for cleavage reactions of E10-32 with wild-type OGT (top) and OGT (D554N) (bottom) showing A, B, C and [A+GlcNAc].}
	\label{fig:d554n-cleave-fast}
\end{figure}
%
Next, I tested the ability of OGT (D554N) to cleave the E10-32 peptide; the reaction was complete within 100 minutes, and we did not detect any glycopeptide byproduct (Figure~\ref{fig:d554n-cleave-fast}). I conducted several time-course experiments using stoichiometric, and excess quantities of OGT (D554N) (Figures~\ref{fig:app-TC1-D554N-E10}, \ref{fig:app-TC1-D554N-E10-graphs}, \ref{fig:app-TC2-D554N-E10}, \ref{fig:app-TC2-D554N-E10-graphs}, \ref{fig:app-TC3-D554N-E10}, \ref{fig:app-TC3-D554N-E10-graphs}, and \ref{fig:app-TC4-D554N-E10-graphs}).\par
%
I found that the starting material was almost entirely consumed within the first three minutes of the reaction (compare Figures \ref{fig:app-TC1-WT-E10} and \ref{fig:d554n-3min}), and two intermediates had accumulated to high levels. One of these had the same retention time and exact mass as the glycopeptide intermediate identified in the wild-type OGT cleavage reaction (Figure~\ref{fig:glycopeptides}). The other had an \textit{m/z} of 840.8784 (4+), corresponding to loss of water from the starting material (Figure~\ref{fig:d554n-3min}).\par
%
\begin{figure}[ht] %shows a single 3 min time point
	\centerline{\includegraphics[width=0.45\columnwidth]{mechanism/D554N-3min.eps}}
	\caption[OGT (D554N) rapidly forms two intermediates]{OGT (D554N) rapidly forms two intermediates. An LC trace showing a 3 minute time point for cleavage of E10-32 using OGT (D554N). Peaks for A, B, C, the [A+GlcNAc] intermediate, and another intermediate, [A-H\textsubscript{2}O], are shown. The ``*'' denotes a second peak having the same \textit{m/z} as the [A-H\textsubscript{2}O] species. A complete time course is shown in Figure \ref{fig:app-TC1-D554N-E10}.}
	\label{fig:d554n-3min}
\end{figure}
%
The dramatic increase in the abundance of the glycopeptide intermediate, together with elimination of the byproduct glycopeptide species, suggested that OGT (D554N) would be a useful tool to study the mechanism of HCF-1 cleavage. In order to be confident that wild-type OGT and OGT (D554N) were effecting cleavage by the same mechanism, I tested whether the two enzymes had the same cleavage requirements, and produced the same products. Indeed, both enzymes produced products B and C (with the same LC retention times), and required UDP-GlcNAc and the glutamate at position 10 in the HCF-1 sequence for cleavage to occur (Figure \ref{fig:cleavage-requirements}). As such, I used OGT (D554N) for the production and study of intermediates 1 and 2.\par
%
\begin{figure}%[ht]
	\centerline{\includegraphics[width=0.75\columnwidth]{mechanism/SI-Fig4.eps}}
	\caption[OGT (D554N) and wild-type have the same substrate requirements for HCF-1 cleavage]{OGT (D554N) and wild-type have the same substrate requirements for HCF-1 cleavage. SDS-PAGE analysis detected with Coomassie showing substrate requirements for cleavage of HCF-3R constructs (see Table \ref{tab:listofpeptides}). The ``X'' in HCF-3R-XAA, is the identity of residue 10 in the first repeat, either Glu (E10) or Asp (D10); UDP and \textit{5S} denote the use of UDP and UDP-\textit{5S}GlcNAc, respectively, rather than UDP-GlcNAc in the reaction. These data show that cleavage occurs when UDP-GlcNAc, but not UDP or UDP-\textit{5S}GlcNAc~\cite{Lazarus:2012gw}, is used and that the repeat must contain glutamic acid, rather than aspartate, at position 10.}
	\label{fig:cleavage-requirements}
\end{figure}
%
We observed the same anhydropeptide species (intermediate 2) in cleavage reactions with wild-type enzyme (Figure~\ref{fig:wt-int-2-formation}), but it accumulated to lower levels (compare Figure~\ref{fig:app-TC1-WT-E10} and Figure~\ref{fig:app-TC1-D554N-E10}).\par
%
\begin{figure}[ht]
	\centerline{\includegraphics[width=0.55\columnwidth]{mechanism/Wt-int2-plot.eps}}
	\caption[Wild-type OGT produces an anhydropeptide intermediate (intermediate 2)]{Wild-type OGT produces an anhydropeptide intermediate (intermediate 2). Integration of the EIC for Int-2 (intermediate 2) (\textit{m/z} 840.8784; 4+) (data shown in Figure~\ref{fig:app-TC1-WT-E10}). Integration for Int-2 included both peaks. Values were corrected based on the signal of the EIC from a blank run.}
	\label{fig:wt-int-2-formation}
\end{figure}
%
\section{Characterizing the anhydropeptide intermediate} \label{AP-characterization}
%
Since the anhydropeptide ([M-H\textsubscript{2}O]) intermediate appeared as two peaks, I compared their MS/MS fragmentation patterns and, except for minor differences in fragment ion intensities, they were identical (Figure~\ref{fig:int-2-peaks-same}).\par
%
\begin{figure}[ht]
	\centerline{\includegraphics[width=1\columnwidth]{mechanism/int2-peak-differences.eps}}
	\caption[Both peaks of Intermediate 2 have the same MS/MS fragmentation pattern]{Both peaks of Intermediate 2 have the same MS/MS fragmentation pattern. Comparison of the CID MS/MS spectra for the two peaks present in the Int-2 (intermediate 2) (\textit{m/z} 840.8784; 4+) EIC. Both by CID (shown) and HCD, the fragmentation patterns are essentially indistinguishable other than slight differences in relative abundance for some fragment ions.}
	\label{fig:int-2-peaks-same}
\end{figure}
%
The fragmentation data localized the dehydration to the C\textsuperscript{9}E\textsuperscript{10} region of the peptide, rather than the C-terminal threonine-rich region, which is also prone to dehydration during the CID/HCD fragmentation process~\cite{Ballard:1993hg} (see Figures \ref{fig:int-2-HCD-spectrum} and \ref{fig:dehydration-not-cterm}, and Tables \ref{tab:y-ion-table-HCD-Int-2}, \ref{tab:b-ion-table1-HCD-int-2}, \ref{tab:b-ion-table2-HCD-int-2}, \ref{tab:y-ion-table-CID-Int-2}, \ref{tab:b-ion-table-CID-int-2}, \ref{tab:b-ion-table-CID-int-2-1}, \ref{tab:y-ion-table-HCD-A}, \ref{tab:b-ion-table1-HCD-A}, \ref{tab:b-ion-table2-HCD-A}). These LC-MS/MS data for the anhydropeptide species were consistent with formation of an internal pyroglutamate (as indicated by the red arrow in Figure~\ref{fig:mechanism-hcf-proposed-GP-intermediate}), but we were not sure why there were two peaks.\par
%
\begin{figure}[ht]
	\centerline{\includegraphics[width=0.85\columnwidth]{mechanism/SI-Fig13.eps}}
	\caption[HCD MS/MS spectrum of Int-2]{HCD MS/MS spectrum of Int-2. \textit{b} and \textit{y} ions obtained by HCD fragmentation are shown for major peaks. Not all peaks are labeled for clarity. Observed ions for Int-2, both by CID and HCD, are provided in Tables \ref{tab:y-ion-table-HCD-Int-2}, \ref{tab:b-ion-table1-HCD-int-2}, \ref{tab:b-ion-table2-HCD-int-2}, \ref{tab:y-ion-table-CID-Int-2}, \ref{tab:b-ion-table-CID-int-2}, \ref{tab:b-ion-table-CID-int-2-1}. Some ions derived from ammonia or water loss are also not annotated. The top spectrum is the full MS/MS spectrum, and the bottom spectrum is a zoom of the region from about 550--900 \textit{m/z}, as indicated by the blue bar. Based on the collective fragmentation data, we mapped the site of dehydration to the C\textsuperscript{9}E\textsuperscript{10} region of the peptide (HCF repeat numbering).}
	\label{fig:int-2-HCD-spectrum}
\end{figure}
%
%
\begin{sidewaystable} %Y ions Int-2 HCD (Supp Table 1)
    \begin{center}
        \begin{tabular}{|c|c|c|c|c|c|}
        \hline
        Residue & C-term. & \textit{y}\textsuperscript{+} obs. (\textit{m/z})$\dagger$ & (\textit{y}-H\textsubscript{2}O)\textsuperscript{+} obs. (\textit{m/z})$\dagger$ & \textit{y}\textsuperscript{2+} obs. (\textit{m/z})$\dagger$ & (\textit{y}-H\textsubscript{2}O)\textsuperscript{2+} obs. (\textit{m/z})$\dagger$\\
        \hline
        H(amidated) & 1 	& 155.0926 	& -- 		 & --		& -- 		\\ \hline
        Q 			& 2 	& 283.1510 	& -- 		 & --		& -- 		\\ \hline
        G 			& 3 	& 340.1724 	& -- 		 & --		& -- 		\\ \hline
        A 			& 4 	& 411.2095 	& -- 		 & --		& -- 		\\ \hline
        M 			& 5 	& 542.2503 	& -- 		 & --		& -- 		\\ \hline
        N 			& 6 	& 656.2925 	& -- 		 & --		& -- 		\\ \hline
        S 			& 7 	& 743.3261 	& -- 		 & --		& -- 		\\ \hline
		T 			& 8 	& 844.3714 	& -- 		 & 422.6895	& 413.6846 	\\ \hline
		A 			& 9 	& 915.4088 	& 897.3971 	 & --		& 449.2028 	\\ \hline
		T 			& 10 	& 1016.4529 & 998.4398 	 & --		& 499.7266 	\\ \hline
		T 			& 11 	& 1117.5004 & 1099.4962  & --		& 550.2511 	\\ \hline
        \end{tabular}
        \caption[List of \textit{y} ions for Int-2 by HCD]{List of \textit{y} ions for Int-2 by HCD. $\dagger$~Errors are shown in Figure~\ref{fig:MSMS-error-plot}.}
        \label{tab:y-ion-table-HCD-Int-2}
    \end{center}
\end{sidewaystable}
%
%
\begin{table} %B ions Int-2 HCD (1/2) (Supp Table 2 (1/2))
    \begin{center}
        \begin{tabular}{|c|c|c|c|}
        \hline
        Residue & N-term. & \textit{b}\textsuperscript{+} obs. (\textit{m/z})$\dagger$ & (\textit{b}-H\textsubscript{2}O)\textsuperscript{3+} obs. (\textit{m/z})$\dagger$\\
        \hline
        Y 	& 1 		& -- 		& -- 		\\ \hline
        V 	& 2 		& 263.1386 	& -- 		\\ \hline
        R 	& 3 		& 419.2395 	& -- 		\\ \hline
        V 	& 4 		& 518.3076 	& -- 		\\ \hline
        C 	& 5 		& -- 		& -- 		\\ \hline
        S 	& 6 		& -- 		& -- 		\\ \hline
        N 	& 7 		& 822.3903 	& -- 		\\ \hline
		P 	& 8 		& -- 		& -- 		\\ \hline
		P 	& 9 		& -- 		& -- 		\\ \hline \hline
		N   & 19        & -- 		& --		\\ \hline
		T 	& 20	 	& -- 		& 724.6581 	\\ \hline
		A 	& 21	 	& -- 		& -- 		\\ \hline
       	\end{tabular}
        \caption[List of singly- and triply-charged \textit{b} ions for Int-2 by HCD]{List of singly- and triply-charged \textit{b} ions for Int-2 by HCD. $\dagger$~Errors are shown in Figure~\ref{fig:MSMS-error-plot}. The double horizontal line marks a break in sequence numbering.}
        \label{tab:b-ion-table1-HCD-int-2}
    \end{center}
\end{table}
%
%
\begin{table} %B ions Int-2 HCD (2/2) (Supp Table 2 (2/2))
    \begin{center}
        \begin{tabular}{|c|c|c|c|}
        \hline
        Residue & N-term. & (\textit{b}-H\textsubscript{2}O)\textsuperscript{2+} obs. (\textit{m/z})$\dagger$ & (\textit{b}-2H\textsubscript{2}O)\textsuperscript{2+} obs. (\textit{m/z})$\dagger$\\
        \hline
        C 	& 10 		& -- 			& -- 		\\ \hline
        E 	& 11 		& 615.7719 		& -- 		\\ \hline
        T 	& 12 		& 666.2944 		& -- 		\\ \hline
        H 	& 13 		& 734.8252 		& -- 		\\ \hline
        Q 	& 14 		& 798.8547 		& -- 		\\ \hline
        T 	& 15 		& 849.3768 		& 840.3713 	\\ \hline
        G 	& 16 		& 877.8874 		& 868.8799 	\\ \hline
		T 	& 17 		& 928.4115 		& 919.4068 	\\ \hline
		T 	& 18 		& 978.9337 		& 969.9290 	\\ \hline
		N   & 19        & 1035.9566 	& 1026.9483	\\ \hline
		T 	& 20	 	& 1086.4812 	& 1077.4728	\\ \hline
		A	& 21	 	& 1121.9928 	& -- 		\\ \hline
       	\end{tabular}
        \caption[List of doubly-charged \textit{b} ions for Int-2 by HCD]{List of doubly-charged \textit{b} ions for Int-2 by HCD. $\dagger$~Errors are shown in Figure~\ref{fig:MSMS-error-plot}.}
        \label{tab:b-ion-table2-HCD-int-2}
    \end{center}
\end{table}
%
%
\begin{sidewaystable} %Y ions Int-2 CID (Supp Table 3)			
    \begin{center}
        \begin{tabular}{|c|c|c|c|c|}
        \hline
        Residue & C-term. & \textit{y}\textsuperscript{+} obs. (\textit{m/z})$\dagger$ & \textit{y}\textsuperscript{2+} obs. (\textit{m/z})$\dagger$ & (\textit{y}-H\textsubscript{2}O)\textsuperscript{2+} obs. (\textit{m/z})$\dagger$\\
        \hline
        H(amidated) & 1 	& --	 	& -- 		 & --		\\ \hline
        Q 			& 2 	& --	 	& -- 		 & --		\\ \hline
        G 			& 3 	& 340.1720 	& -- 		 & --		\\ \hline
        A 			& 4 	& 411.2090 	& -- 		 & --		\\ \hline
        M 			& 5 	& 542.2484 	& -- 		 & --		\\ \hline
        N 			& 6 	& 656.2907 	& -- 		 & --		\\ \hline
        S 			& 7 	& 743.3247 	& -- 		 & --		\\ \hline
		T 			& 8 	& 844.3689 	& 422.6908 	 & --		\\ \hline
		A 			& 9 	& 915.4070 	& 458.2063 	 & 449.2025	\\ \hline
		T 			& 10 	& 1016.4545 & 508.7373 	 & 499.7283	\\ \hline
		T 			& 11 	& 1117.5017 & --		 & 550.2511	\\ \hline
		A 			& 12 	& 1188.5368 & --	 	 & --		\\ \hline
		T 			& 13 	& 1289.5869 & --		 & --		\\ \hline
        \end{tabular}
        \caption[List of \textit{y} ions for Int-2 by CID]{List of \textit{y} ions for Int-2 by CID. $\dagger$~Errors are shown in Figure~\ref{fig:MSMS-error-plot}.}
        \label{tab:y-ion-table-CID-Int-2}
    \end{center}
\end{sidewaystable}
%
%
\begin{table} %B ions Int-2 CID (Supp Table 4a)
    \begin{center}
        \begin{tabular}{|c|c|c|c|}
        \hline
        Residue & N-term. & \textit{b}\textsuperscript{+} obs. (\textit{m/z})$\dagger$ & \textit{b}\textsuperscript{2+} obs. (\textit{m/z})$\dagger$\\
        \hline
        C 	& 5 		& -- 			& -- \\ \hline
        S 	& 6 		& 708.3482		& -- \\ \hline
        N 	& 7 		& 822.3897		& 411.7006 \\ \hline
        P 	& 8 		& --		 	& 460.2247 \\ \hline
        P 	& 9 		& --		 	& -- \\ \hline
        C 	& 10 		& --		 	& 560.2535 \\ \hline
%        \hline \hline
       	\end{tabular}
        \caption[List of \textit{b} ions for Int-2 by CID]{List of \textit{b} ions for Int-2 by CID. $\dagger$~Errors are shown in Figure \ref{fig:MSMS-error-plot}.}
        \label{tab:b-ion-table-CID-int-2}
    \end{center}
\end{table}
%
%
\begin{table} %B ions Int-2 CID (2/2) (Supp Table 4b)
    \begin{center}
        \begin{tabular}{|c|c|c|c|}
        \hline
        Residue & N-term. & (\textit{b}-H\textsubscript{2}O)\textsuperscript{+} obs. (\textit{m/z})$\dagger$ & (\textit{b}-H\textsubscript{2}O)\textsuperscript{2+} obs. (\textit{m/z})$\dagger$\\
        \hline
		T 	& 12 		& 1331.5869		& -- \\ \hline
		H 	& 13 		& 1468.6412		& 734.8235 \\ \hline
		Q   & 14        & 1596.6996 	& 798.8551 \\ \hline
		T 	& 15	 	& 1697.7511		& 849.3767 \\ \hline
		G	& 16	 	& -- 			& 877.8901 \\ \hline
		T 	& 17 		& --			& 928.4123 \\ \hline
        T 	& 18 		& --			& 978.9359 \\ \hline
        N 	& 19 		& --			& 1035.9563 \\ \hline
		T 	& 20 		& --			& 1086.4824 \\ \hline
		A 	& 21 		& --			& 1121.9988 \\ \hline
		T   & 22        & -- 			& 1172.53 \\ \hline
		T	& 23	 	& -- 			& 1223.05 \\ \hline
		A	& 24	 	& -- 			& 1258.5627 \\ \hline
       	\end{tabular}
        \caption[List of dehydrated \textit{b} ions for Int-2 by CID]{List of dehydrated \textit{b} ions for Int-2 by CID. $\dagger$~Errors are shown in Figure \ref{fig:MSMS-error-plot}.}
        \label{tab:b-ion-table-CID-int-2-1}
    \end{center}
\end{table}
%
%
\begin{sidewaystable} %Y ions E10-32 HCD (Supp Table 5)			
    \begin{center}
        \begin{tabular}{|c|c|c|c|c|c|}
        \hline
        Residue & C-term. & \textit{y}\textsuperscript{+} obs. (\textit{m/z})$\dagger$ & \textit{y}\textsuperscript{2+} obs. (\textit{m/z})$\dagger$ & (\textit{y}-H\textsubscript{2}O)\textsuperscript{+} obs. (\textit{m/z})$\dagger$ & (\textit{y}-H\textsubscript{2}O)\textsuperscript{2+} obs. (\textit{m/z})$\dagger$\\
        \hline
        H(amidated) & 1 	& 155.0928	& -- 		 &	--			& --		\\ \hline
        Q 			& 2 	& --	 	& -- 		 &	--			& --		\\ \hline
        G 			& 3 	& 340.1729 	& -- 		 &	--			& --		\\ \hline
        A 			& 4 	& 411.2099 	& -- 		 &	--			& --		\\ \hline
        M 			& 5 	& 542.2510 	& -- 		 &	--			& --		\\ \hline
        N 			& 6 	& 656.2933 	& -- 		 &	--			& --		\\ \hline
        S 			& 7 	& 743.3254 	& -- 		 &	--			& 363.1610	\\ \hline
		T 			& 8 	& 844.3726 	& 422.6898 	 &	826.3614	& 413.6851	\\ \hline
		A 			& 9 	& 915.4108 	& 458.2095 	 &	897.3971	& 449.2036	\\ \hline
		T 			& 10 	& 1016.4541 & --	 	 &	998.4438	& 499.7275	\\ \hline
		T 			& 11 	& 1117.5040 & --		 &	1099.4923	& 550.2521	\\ \hline
		A 			& 12 	& --		& --	 	 &	--			& --		\\ \hline
        \end{tabular}
        \caption[List of \textit{y} ions for E10-32 by HCD]{List of \textit{y} ions for E10-32 by HCD $\dagger$~Errors are shown in Figure~\ref{fig:MSMS-error-plot}.}
        \label{tab:y-ion-table-HCD-A}
    \end{center}
\end{sidewaystable}
%
\begin{table} %B ions E10-32 HCD (1/2) (Supp Table 6)
    \begin{center}
        \begin{tabular}{|c|c|c|c|}
        \hline
        Residue & N-term. & \textit{b}\textsuperscript{+} obs. (\textit{m/z})$\dagger$ & \textit{b}\textsuperscript{2+} obs. (\textit{m/z})$\dagger$\\
        \hline
        V 	& 2 		& 263.1392					& --	 											\\ \hline
        R 	& 3 		& 419.2400					& --	 											\\ \hline
        V 	& 4 		& 518.3087					& -- 												\\ \hline
        C 	& 5 		& 621.3179 					& -- 												\\ \hline
        S 	& 6 		& 708.3492					& -- 												\\ \hline
        N 	& 7 		& 822.3923					& 411.7000 	(402.6949)\textsuperscript{$\ddagger$} 	\\ \hline
        P 	& 8 		& --		 				& 460.2264 	(451.2238)\textsuperscript{$\ddagger$} 	\\ \hline
        P 	& 9 		& --		 				& 508.7544 											\\ \hline
        C 	& 10 		& --		 				& 560.2573 											\\ \hline
        E   & 11 		& --						& 624.7779											\\ \hline
		T 	& 12 		& --						& 675.3015											\\ \hline
		H 	& 13 		& --						& 743.8321	(734.8258)\textsuperscript{$\ddagger$} 	\\ \hline
		Q   & 14        & --	 					& 807.8615	(798.8545)\textsuperscript{$\ddagger$}	\\ \hline
		T 	& 15	 	& --						& 858.3858	(849.3779)\textsuperscript{$\ddagger$}	\\ \hline
		G	& 16	 	& -- 						& 886.8935	(877.8891)\textsuperscript{$\ddagger$}	\\ \hline
		T 	& 17 		& --						& 937.4193	(928.4130)\textsuperscript{$\ddagger$}	\\ \hline
        T 	& 18 		& --						& 987.9402	(978.9350)\textsuperscript{$\ddagger$}	\\ \hline
        N 	& 19 		& --						& 1044.9629	(1035.9553)\textsuperscript{$\ddagger$}	\\ \hline
		T 	& 20 		& --						& 1095.4865	(1086.4810)\textsuperscript{$\ddagger$}	\\ \hline
       	\end{tabular}
        \caption[List of \textit{b} ions for E10-32 by HCD]{List of \textit{b} ions for E10-32 by HCD. $\dagger$~Errors are shown in Figure~\ref{fig:MSMS-error-plot}. \textsuperscript{$\ddagger$}~Denotes (\textit{b}-H\textsubscript{2}O)\textsuperscript{2+} ions also observed.}
        \label{tab:b-ion-table1-HCD-A}
    \end{center}
\end{table}
%
\begin{table} %B ions E10-32 HCD (2/2) (Supp Table 6)
    \begin{center}
        \begin{tabular}{|c|c|c|c|c|}
        \hline
        Residue & N-term. & \textit{b}\textsuperscript{2+} obs. (\textit{m/z})$\dagger$ & (\textit{b}-2H\textsubscript{2}O)\textsuperscript{2+} obs. (\textit{m/z})$\dagger$ & \textit{b}\textsuperscript{3+} obs. (\textit{m/z})$\dagger$\\
        \hline
        T 	& 18 		& --						& 969.9297	& --		\\ \hline
        N 	& 19 		& --						& --		& --		\\ \hline
		T 	& 20 		& --						& --		& 730.6610 (724.6583)\textsuperscript{$\ddagger$}	\\ \hline
		A 	& 21 		& 1131.0024					& --		& --		\\ \hline
       	\end{tabular}
        \caption[List of additional \textit{b} ions for E10-32 by HCD]{List of additional \textit{b} ions for E10-32 by HCD. $\dagger$~Errors are shown in Figure~\ref{fig:MSMS-error-plot}. \textsuperscript{$\ddagger$}~Denotes (\textit{b}-H\textsubscript{2}O)\textsuperscript{3+} ions.}
        \label{tab:b-ion-table2-HCD-A}
    \end{center}
\end{table}
%
To rule out the possibility that one of the anhydropeptide species was a thiolactone resulting from cysteine participation (Figure~\ref{fig:int-with-without-cys}), which was previously suggested as a mechanistic possibility (Figure~\ref{fig:hcf-proposed-mech} (mechanism 2), and~\cite{Lazarus:2013bq}), I replaced Cys9 in the E10-32 peptide with Arg, an amino acid found in a proteolytic repeat from the \textit{Odobenus rosmarus divergens} (walrus) HCF-1 ortholog (see section~\ref{sec:hcf-C9-muts}). R9E10-32 was a good cleavage substrate (Figure~\ref{fig:c9r-time-course-plots}); we again observed two peaks corresponding to an anhydropeptide intermediate in reactions with both wild-type OGT and OGT (D554N) (Figure~\ref{fig:common-retention-times}). We also observed two anhydropeptide peaks with E10V11-32 (Figure~\ref{fig:common-retention-times}), S9E10-32, and A9E10-32 (data not shown). Taken together, these data exclude the possibility that the second species is the result of cysteine participation. An expanded discussion on the nature of the two anhydropeptide species in provided in section~\vref{sec:why-two-AP}.\par
%
\begin{figure}[ht]
	\centerline{\includegraphics[width=0.85\columnwidth]{mechanism/reproduced-mechanisms-with-without-cys9.eps}}
	\caption[Possible Mechanisms, with or without cysteine participation]{Possible Mechanisms, with or without cysteine participation. After initial glycosylation of the glutamate side chain (see Figure~\ref{fig:hcf-proposed-mech}), we reason that formation of the requisite internal pyroglutamate can occur either in a Cys-independent or a Cys-dependent manner. In the first case, the activated glutamate side chain is trapped by the C--E amide nitrogen to form an internal pyroglutamate directly. We also consider the case where the ester is trapped by the thiol of Cys to form a macrocyclic thiolactone. Such a species would potentially be in equilibrium with an internal pyroglutamate~\cite{Erickson:1983uv,Khan:1982wa,Khan:1984fd,Khan:1986tb}. In both cases hydrolysis of the internal pyroglutamate leads to product formation.}
	\label{fig:int-with-without-cys}
\end{figure}
%
\begin{figure}[ht]
	\centerline{\includegraphics[width=0.85\columnwidth]{mechanism/SI-Fig16.eps}}
	\caption[Plots of R9E10-32 cleavage time course with excess of OGT (D554N)]{Plots of R9E10-32 cleavage time course with excess of OGT (D554N). Integration of individual EICs provided quantitation of response units for each species at each time. Starting material, [R9E10-32] (both charge states,\textit{m/z} 858.4048 (4+) and 686.9253 (5+), were integrated), decays analogously to E10-32. A glycopeptide (\textit{m/z} 727.5412 (5+)) forms rapidly, and subsequently decays as two anhydropeptide species (\textit{m/z} 683.3232 (5+)) form. Both of these species decay as the reaction progresses and products form (N-terminus, \textit{m/z} 397.5415 (3+) and C-terminus, \textit{m/z} 747.6698 (3+); this is the same C-terminal product as E10-32). All values used are corrected based on the signal of an EIC from a blank run.}
	\label{fig:c9r-time-course-plots}
\end{figure}
%
\section{Establishing the temporal sequence for intermediate \\ formation}
%
Having conducted several time courses under conditions where OGT was in excess of the peptide substrate (single-turnover conditions, STO), I was able to unequivocally establish the temporal sequence of events for HCF-1 cleavage. Within 60 seconds the starting material had converted to the glycopeptide intermediate. As this species decayed, one anhydropeptide species appeared (Figure~\ref{fig:temporal-sequence}; see also Figures~\ref{fig:app-TC2-D554N-E10} and~\ref{fig:app-TC2-D554N-E10-graphs}), and the second grew in as the reaction progressed. Both species were consumed by the end of the reaction, indicating that both are on-pathway to cleavage products. Hence, the reaction involves initial formation of a glycopeptide intermediate (Intermediate 1) followed by conversion to an internal pyroglutamate (Intermediate 2) and then backbone cleavage. I measured a rate constant of 0.08 s$^{-1}$ for the consumption of A (starting material) by OGT (D554N), which is approximately two orders of magnitude faster than that of wild-type OGT (compare Figures \ref{fig:cleavage-reaction-plots} and \ref{fig:app-TC2-D554N-E10-graphs}). Hence, replacement of aspartate for asparagine at position 554 dramatically accelerated formation of the glycopeptide intermediate. Overall cleavage rates for wild-type OGT and OGT (D554N) differed only by a factor of two to three, however, evidently because hydrolysis of the internal pyroglutamate is rate determining.\par
%
\begin{figure}[ht]
	\centerline{\includegraphics[width=0.55\columnwidth]{mechanism/temporal-sequence-plot.eps}}
	\caption[Temporal sequence of reaction intermediates]{Temporal sequence of reaction intermediates. The temporal sequence of events was established from a time course of cleavage using a large excess of OGT (D554N). Mole fractions of A (starting material), [A+GlcNAc] and [A-H\textsubscript{2}O], were plotted as a function of time. The data were truncated at 420 seconds, such that products represent a negligible mole fraction. Mole fractions were determined based on the maximal signal for each species, normalized over all species present at that time point.}
	\label{fig:temporal-sequence}
\end{figure}
%
\section{Characterizing the glycopeptide intermediate}
%
Because the use of OGT (D554N) results in a substantial accumulation of the glycopeptide intermediate at early time points, I was able to obtain sufficient material for MS/MS analysis by quenching OGT (D554N) cleavage reactions after about one minute. The intermediate proved stable enough to allow for LC purification, and fragmentation by electron-capture dissociation (ECD) allowed unambiguous assignment of the GlcNAc modification to glutamate 10 (Figures~\ref{fig:glycopeptide-int-ECD}, \ref{fig:ECD-spectrum-int1-zooms}, and \ref{fig:ECD-spectrum-int1-site} and Tables~\ref{tab:c-ion-table1-ECD-Int1}, \ref{tab:c-ion-table2-ECD-Int1}, \ref{tab:z-ion-table1-ECD-Int1}, and \ref{tab:z-ion-table2-ECD-Int1}).\par
%
While the stability of the glycosylated intermediate was unexpected, aspartate- and glutamate-linked glycosyl esters produced by protein ADP-ribosylation are, at least in some cases, also sufficiently stable to allow for isolation and characterization~\cite{Hassa:2006iz,Tao:2009cd}.\par
%
\begin{figure}[ht]
	\centerline{\includegraphics[width=1\columnwidth]{mechanism/ECD-glycopeptide-int.eps}}
	\caption[ECD MS/MS spectrum of Intermediate 1]{ECD MS/MS spectrum of Intermediate 1. Intermediate 1 is a glutamate-linked glycopeptide. Insets show zooms of ions \textit{c}\textsubscript{11}\textsuperscript{+} and \textit{z}\textsubscript{22}\textsuperscript{2+} that flank the glycosylated residue. Additional spectra are presented in Figures~\ref{fig:ECD-spectrum-int1-zooms} and~\ref{fig:ECD-spectrum-int1-site}.}
	\label{fig:glycopeptide-int-ECD}
\end{figure}
%
\begin{table} %C ions Int-1 ECD (1/2) (Supp Table 7)
    \begin{center}
        \begin{tabular}{|c|c|c|c|}
        \hline
        Residue & N-term. & \textit{c}\textsuperscript{+} obs. (\textit{m/z})$\dagger$ & (\textit{c}-1)\textsuperscript{+} obs. (\textit{m/z})$\dagger$ \\
        \hline
		Y	& 1		&	--						&	180.0869			\\ \hline	
		V	& 2		&	--						&	279.1579			\\ \hline	
		R	& 3		&	436.2665				&	435.2585			\\ \hline	
		V	& 4		&	535.3350				&	534.3274			\\ \hline	
		C	& 5		&	638.3452				&	637.3369			\\ \hline	
		S	& 6		&	725.3767				&	724.3687			\\ \hline	
		N	& 7		&	\cellcolor{gray}		& 	\cellcolor{gray}	\\ \hline
		P	& 8		&	\cellcolor{gray}		& 	\cellcolor{gray}	\\ \hline
		P	& 9		&	1033.5258				&	1032.5172			\\ \hline
		C	& 10	&	1136.5349				&	1135.5292			\\ \hline
		E*	& 11	&	1468.6550				&	1467.6505			\\ \hline
		T	& 12	&	1569.7027				&	1568.6995			\\ \hline
		H	& 13	&	1706.7614				&	--					\\ \hline
		Q	& 14	&	--						&	--					\\ \hline
		T	& 15	&	--						&	--					\\ \hline
		G	& 16 	&	--						&	--					\\ \hline
       	\end{tabular}
        \caption[List of singly-charged \textit{c} ions for Intermediate-1 by ECD]{List of singly-charged \textit{c} ions for Int-1 by ECD. *~Denotes a glycosylated glutamic acid residue (E* = E+GlcNAc). $\dagger$~Errors are shown in Figure~\ref{fig:MSMS-error-plot}. Grayed out cells denote ions not observed in ECD fragmentation.}
        \label{tab:c-ion-table1-ECD-Int1}
    \end{center}
\end{table}
%
%
\begin{table} %C ions Int-1 ECD (2/2) (Supp Table 7)
    \begin{center}
        \begin{tabular}{|c|c|c|c|}
        \hline
        Residue & N-term. & \textit{c}\textsuperscript{2+} obs. (\textit{m/z})$\dagger$ & (\textit{c}-1)\textsuperscript{2+} obs. (\textit{m/z})$\dagger$ \\
        \hline				
		H			& 13	&	--						&	853.3816			\\ \hline
		Q			& 14	&	--						&	917.4110			\\ \hline
		T			& 15	&	--						&	967.9349			\\ \hline
		G			& 16 	&	--						&	Occluded			\\ \hline
		T			& 17	&	--						&	1046.9699			\\ \hline
		T			& 18 	&	--						&	1097.4938			\\ \hline
		N			& 19	&	--						&	1154.5155			\\ \hline
		T			& 20 	&	1205.5424				&	--					\\ \hline
		A			& 21	&	--						&	1240.5735			\\ \hline
		T			& 22 	&	--						&	1291.0829			\\ \hline
		T			& 23	&	--						&	1341.6088			\\ \hline
		A			& 24 	&	--						&	1377.1273			\\ \hline
		T			& 25	&	1427.6520				&	--					\\ \hline
		S			& 26 	&	1471.6694				&	--					\\ \hline
		N			& 27 	&	1528.6907				&	--					\\ \hline
		M			& 28	&	1594.2122				&	--					\\ \hline
		A			& 29 	&	1629.7262				&	--					\\ \hline
		G			& 30	&	1658.2428				&	--					\\ \hline
		Q			& 31 	&	1722.2699				&	--					\\ \hline
       	\end{tabular}
        \caption[List of multiply-charged \textit{c} ions for Int-1 by ECD]{List of multiply-charged \textit{c} ions for Int-1 by ECD. $\dagger$~Errors are shown in Figure~\ref{fig:MSMS-error-plot}.}
        \label{tab:c-ion-table2-ECD-Int1}
    \end{center}
\end{table}
%
%
\begin{table} %Z ions Int-1 ECD (1/2) (Supp Table 8)
    \begin{center}
        \begin{tabular}{|c|c|c|c|}
        \hline
        Residue & C-term. & \textit{z}\textsuperscript{+} obs. (\textit{m/z})$\dagger$ & (\textit{z}+1)\textsuperscript{+} obs. (\textit{m/z})$\dagger$ \\
        \hline				
		Q	& 19		&	--				&	1876.8520	\\ \hline
		T	& 18		&	1747.7835		&	1748.7915	\\ \hline
		G	& 17		&	1646.7325		&	1647.7439	\\ \hline
		T	& 16		&	--				&	--			\\ \hline
		T	& 15	 	&	--				&	1489.6792	\\ \hline
		N	& 14		&	1387.6171		&	1388.6247	\\ \hline
		T	& 13	 	&	1273.5738		&	1274.5815	\\ \hline
		A	& 12		&	--				&	--			\\ \hline
		T	& 11	 	&	--				&	--			\\ \hline
		T	& 10		&	1000.4400		&	1001.4460	\\ \hline
		A	& 9 		&	899.3884		&	900.4018	\\ \hline
		T	& 8			&	828.3543		&	829.3626	\\ \hline
		S	& 7	 		&	727.3067		&	728.3149	\\ \hline
		N	& 6			&	640.2746		&	--			\\ \hline
		M	& 5	 		&	526.2315		&	527.2339	\\ \hline
		A	& 4	 		&	395.1909		&	396.1989	\\ \hline
		G	& 3			&	324.1538		&	325.1616	\\ \hline
		Q	& 2	 		&	267.1324		&	268.1402	\\ \hline
		H(amidated)	& 1			&	139.0739		&	140.0817	\\ \hline
       	\end{tabular}
        \caption[List of singly-charged \textit{z} ions for Int-1 by ECD]{List of singly-charged \textit{z} ions for Int-1 by ECD. $\dagger$~Errors are shown in Figure~\ref{fig:MSMS-error-plot}.}
        \label{tab:z-ion-table1-ECD-Int1}
    \end{center}
\end{table}
%
%
\begin{table} %Z ions Int-1 ECD (2/2) (Supp Table 8)
    \begin{center}
        \begin{tabular}{|c|c|c|c|}
        \hline
        Residue & N-term. & \textit{z}\textsuperscript{2+} obs. (\textit{m/z})$\dagger$ & (\textit{z}+1)\textsuperscript{2+} obs. (\textit{m/z})$\dagger$ \\
        \hline
		N	& 26	&	1429.1156				& 	--					\\ \hline
		P	& 25	&	\cellcolor{gray}		& 	\cellcolor{gray}	\\ \hline
		P	& 24	&	\cellcolor{gray}		&	\cellcolor{gray}	\\ \hline
		C	& 23	&	--						&	--					\\ \hline
		E*	& 22	&	--						&	1224.0417			\\ \hline
		T	& 21	&	--						&	1057.9808			\\ \hline
		H	& 20	&	--						&	1007.4568			\\ \hline
       	\end{tabular}
        \caption[List of doubly-charged \textit{z} ions for Int-1 by ECD]{List of doubly-charged \textit{z} ions for Int-1 by ECD. *~Denotes a glycosylated glutamic acid residue (E* = E+GlcNAc). $\dagger$~Errors are shown in Figure~\ref{fig:MSMS-error-plot}. Grayed out cells denote ions not observed in ECD fragmentation.}
        \label{tab:z-ion-table2-ECD-Int1}
    \end{center}
\end{table}
%
\section{Mechanism of HCF-1 cleavage by OGT} \label{sec:cleavage-mechanism}
%
These studies show that OGT effects HCF-1 amide bond cleavage by the mechanism in Figure~\ref{fig:ch3-cleavage-mechanism}. Glycosylation on glutamate is followed by conversion to an internal pyroglutamate, which is susceptible to spontaneous hydrolysis~\cite{Erickson:1983uv,Khan:1982wa,Khan:1984fd,Khan:1986tb,Nalbone:2016fw}. Because an internal pyroglutamate can form non-enzymatically if a glutamate side chain is activated~\cite{Nalbone:2016fw}, we wondered whether Intermediate 1 converts to Intermediate 2 on or off OGT.\par
%
In order to test this, I isolated the glycopeptide intermediate (Intermediate 1) and re-subjected it to the reaction conditions, but without OGT. In order to differentiate newly formed products from those that may have formed during the isolation process, I used \textsuperscript{18}O-water for reactions conducted with isolated glycopeptide. When \textsuperscript{18}O-water is used as the reaction medium, the incorporation of a single \textsuperscript{18}O atom into the C-terminal Cys residue of the N-terminal cleavage product is observed. I observed formation of an anhydropeptide followed by hydrolytic cleavage over 90 minutes. Because the rate of conversion of glycopeptide to internal pyroglutamate was an order of magnitude slower than observed in the presence of OGT (Figure~\ref{fig:on-or-off-enzyme}), we conclude that both the first and second steps shown in Figure~\ref{fig:ch3-cleavage-mechanism} occur while the peptide is bound to OGT. Hydrolysis likely occurs after dissociation from the enzyme.\par
%
\begin{figure}
	\centerline{\includegraphics[width=0.7\columnwidth]{mechanism/hcf-cleavage-mechanism.eps}}
	\caption[The mechanism of HCF-1 cleavage proceeds via glycosylation on glutamate followed by decomposition to an internal pyroglutamate, which undergoes backbone hydrolysis]{The mechanism of HCF-1 cleavage proceeds via glycosylation on glutamate followed by decomposition to an internal pyroglutamate, which undergoes backbone hydrolysis. Chemical mechanism of HCF-1 cleavage by OGT. Both glycosylation on glutamate and conversion to the internal pyroglutamate (Intermediate 2) occur on the enzyme (see text and Figure~\ref{fig:on-or-off-enzyme}), but hydrolysis likely occurs after dissociation.}
	\label{fig:ch3-cleavage-mechanism}
\end{figure}
%
\begin{figure}[b]
	\caption[Plots for the reaction of Int-1 without OGT]{\textit{(On the following page)} Plots for the reaction of Int-1 without OGT. Data is based on a time course where Int-1 is incubated in reaction buffer without OGT. (a) Integration of individual EIC traces provides quantitation, in response units, for each species (A, Int-1, Int-2, B and C) at each time. The \textit{m/z} for each species is the same as in previous captions. The amount of starting material (A) (not shown) does not change over the course of the incubation; this material co-purified with the glycopeptide intermediate. There is a distinct lag in product formation, as seen in other time courses, shown in Figures~\ref{fig:app-TC1-D554N-E10-graphs}, \ref{fig:app-TC2-D554N-E10-graphs}, and~\ref{fig:app-TC3-D554N-E10-graphs}. As seen previously, Int-2 forms and then decays, concomitant with the formation of products. (b) The disappearance of Int-1 was fit to a first-order decay (Equation~\ref{first-order-decay}), both in the presence (left) and absence (right) of OGT. Data for Int-1 conversion, in the presence of enzyme, is taken from the 100 minute time course shown in Figure~\ref{fig:app-TC1-D554N-E10} (see also Figure~\ref{fig:app-TC1-D554N-E10-graphs}). The curve is fit from the time point where maximum Int-1 has accumulated.}
	\label{fig:on-or-off-enzyme}
\end{figure}
\begin{figure}[p]
	\centerline{\includegraphics[width=0.95\columnwidth]{mechanism/SI-Fig19.eps}}
\end{figure}
%
\section{OGT (D554N) accelerates the glycosylation step \\ of HCF-1 cleavage}
%
Though OGT (D554N) accelerates the formation of the two cleavage reaction intermediates, the apparent overall reaction rate was similar for wild-type OGT and OGT (D554N). Comparing the rates of E10-32 consumption by wild-type OGT and OGT (D554N), suggested that OGT (D554N) catalyzes glycosylation on glutamate much faster than the wild-type enzyme (Figure~\ref{fig:compare-glyco-rates}).
%
\begin{figure}
	\centerline{\includegraphics[width=0.9\columnwidth]{mechanism/compare_glyco_rates.eps}}
	\caption[Comparing the rate of substrate glycosylation for wild-type OGT and OGT (D554N)]{Comparing the rate of substrate glycosylation for wild-type OGT and OGT (D554N). Data for the rate of starting material consumption by wild-type OGT and OGT (D554N) are reproduced from Figures~\ref{fig:cleavage-reaction-plots} and~\ref{fig:app-TC2-D554N-E10}, respectively.}
	\label{fig:compare-glyco-rates}
\end{figure}
%
This suggests that OGT (D554N) may provide a lower-energy pathway to the glycopeptide intermediate (Int-1)(Figure~\ref{fig:free-energy-coordinate}). Assuming that the free-energy of TS2 and the internal pyroglutamate (Int-2) are relatively unchanged for OGT (D554N), we would expect that accelerating the first step (glycosylation) would lead an accumulation of intermediates 1 and 2, for a process where the initial barrier for the first step is high and the rate-determining step is the conversion of intermediate 2 to products. This does not account for the possibility that dissociation of intermediate 2 from OGT may also be rate determining.\par
%
%\begin{figure}[ht]
%	\centerline{\includegraphics[width=1\columnwidth]{mechanism/free-energy-d554n-1.eps}}
%	\caption[Free energy diagram for HCF-1 cleavage]{Free energy diagram for HCF-1 cleavage. A pictorial representation of free energy vs. reaction coordinate for the steps of HCF-1 cleavage. The vertical axis is not to scale, rather it is intended to aid in qualitative discussions made in the text.}
%	\label{fig:free-energy-coordinate}
%\end{figure}
%
\begin{figure}[htb]
  	\centering
  	\begin{turn}{90}
  		\begin{minipage}{1.0\textwidth}
  		\centering
    	\centerline{\includegraphics[width=1\columnwidth]{mechanism/free-energy-d554n-1.eps}}
  		\caption[Free-energy diagram for HCF-1 cleavage]{Free-energy diagram for HCF-1 cleavage. A pictorial representation of free energy vs. reaction coordinate for the steps of HCF-1 cleavage. The vertical axis ($\Delta$G) is not to scale, and is intended to aid in qualitative discussions made in the text.}
  		\label{fig:free-energy-coordinate}
  		\end{minipage}
  	\end{turn}
\end{figure}
%
It might seem curious that a mutant impaired with respect to Ser/Thr glycosylation (see Figure~\ref{fig:d554n-cleave-not-glyco}) would be improved with respect to glutamate glycosylation. Aspartate 554, however, is proposed to serve as a base to shuttle the proton released from the acceptor hydroxyl out of the active site following glycosylation~\cite{Janetzko:2014fy,Lazarus:2012gw} and this function would dispensable for glycosylation of a carboxylate (see also section~\ref{sec:ogt-muts}). While this explanation resolves a seeming paradox, it does not address \emph{how} OGT (D554N) can catalyze glutamate glycosylation faster than the wild-type enzyme.\par
%
Michael B. Lazarus had previously solved a 2.05 \AA~ternary structure of hOGT4.5 (D554N) bound to UDP and a CKII glycopeptide (Figure~\ref{fig:d554n-xray} and Table~\ref{tab:d554n-xray-parameters}). This structure showed that the D554N mutant protein is virtually identical to the wild-type enzyme (0.36 \AA~root mean squared deviation (RMSD))~\cite{Lazarus:2012gw}, implying that the difference in activity is not due to a conformational change in the protein. Instead, we reasoned that glutamate glycosylation may be disfavored by a repulsive interaction with the D554 side chain in wild-type OGT. In order for glutamate 10 to attack the anomeric carbon of UDP-GlcNAc, it must approach within 4.5 \AA~of the D554 carboxylate (Figure~\ref{fig:close-contact-E10-D554}), and this unfavorable interaction is eliminated in the D554N variant.\par
%
\begin{figure}[b]
	\caption[Mutating Asp554 to asparagine does not affect the structure of the OGT active site]{\textit{(On the following page)} Mutating Asp554 to asparagine does not affect the structure of the OGT active site. (a) Overlay of the UDP:hOGT4.5 (D554N):CKII glycopeptide ternary product complex, with the wild-type ternary product complex (PDB: 4GYW). The glycopeptide and important side chains are shown in stick representation. The wild-type structure is shown in yellow and the D554N structure is shown in cyan. The structures are nearly identical with an RMSD of 0.36 \AA. (b) Electrostatic surface rendering of the wild-type UDP:hOGT4.5:glycoCKII ternary product complex crystal structure. The peptide is shown in cyan and the GlcNAc is shown in yellow. The D554 side chain is shown in ball and stick representation, highlighting the negative charge (red) around that residue. Electrostatic scale is shown in the lower left corner. (c) Electrostatic surface rendering of the UDP:hOGT4.5 (D554N):glycoCKII ternary product complex crystal structure. The peptide is shown in cyan and the GlcNAc in yellow as in (b). The Asn554 side chain is shown and the neutral charge (white) in the vicinity of this side chain is indicated by the white color in the surface rendering.}
	\label{fig:d554n-xray}
\end{figure}
\begin{figure}[p]
	\centerline{\includegraphics[width=0.65\columnwidth]{mechanism/D554_SI_figure_0_7.png}}
\end{figure}
%
\begin{figure}[ht]
	\centerline{\includegraphics[width=0.95\columnwidth]{mechanism/D554_SI_figure_0_8.png}}
	\caption[E10 of the HCF repeat is repelled by Asp554]{E10 of the HCF repeat is repelled by Asp554. Stereoimage of a view from a crystal structure of OGT with a bound nucleotide-sugar analog and an analog of an HCF-1 proteolytic repeat in which E10 is replaced with glutamine (PDB: 4N3B). The peptide and D554 residue are shown in cyan and the UDP-5SGlcNAc is shown in yellow. The side chains of D554 (from OGT) and the E10Q side chain (from the HCF-1 repeat) are 6.4 \AA~away in the crystal structure (as shown). In order for E10 to reach the anomeric carbon of the sugar, the side chain must rotate towards it, which would place it within 4.5 \AA~of the D554 carboxylate. The resulting repulsive interaction is unfavorable and we propose that the D554N mutant glycosylates E10 more rapidly because this electrostatic repulsion is relieved.}
	\label{fig:close-contact-E10-D554}
\end{figure}
%
A repulsive interaction involving the reactive glutamate of the HCF proteolytic repeat and the OGT co-substrate UDP-GlcNAc was previously suggested~\cite{Bhuiyan:2015fg}. Whether Asp554 disfavors glycosylation directly through electrostatic repulsion, or indirectly remains to be precisely determined.\par
%
\section{The Nature of two anhydropeptide species} \label{sec:why-two-AP}
%
For all peptide substrates examined, EIC traces of OGT-formed anhydropeptide showed two distinct peaks. Their relative abundances varied with time, with one forming earlier in the reaction than the other (see Figure~\ref{fig:app-TC2-D554N-E10}). However, both peaks are consumed by the end of the reaction, suggesting that they are both on-pathway intermediates. As mentioned previously in this chapter (see section~\ref{AP-characterization}), MS/MS spectra (both CID and HCD) collected for both peaks showed no unique fragment ions, though several fragments differed slightly in their relative abundance (Figure~\ref{fig:int-2-peaks-same}). This suggested that the two species were likely stereoisomers.\par
%
When Int-1 was isolated and allowed to convert to products (via Int-2), only a \emph{single} anhydropeptide species was observed (corresponding to the anhydropeptide formed second in the presence of enzyme) (Figure~\ref{fig:only-one-AP-without-OGT}). Based on this, we speculate that the anhydropeptide species formed second in the enzymatic reaction is the result of a Int-2a $\rightarrow$ Int-2b process (where a and b denote the species formed first and second, respectively), which occurs after dissociation from OGT. If this process were sufficiently fast, we would not expect to observe Int-2a in the absence of enzyme. We reasoned that there were two possibilities for the difference between Int-2a and Int-2b: (1) they were rotational isomers about the internal pyroglutamate bond, analogous to that seen for some proline-containing peptides~\cite{Meyer2013}; (2) they were diastereomers~\cite{GoddardBorger:2016eza}. Attempts to isolate each of these species individually (using an approach analogous to the isolation of Int-1), to test whether they were able to interconvert, or whether only a single one led directly to products, were fraught with difficulty, and sufficient quantities of material have yet been unobtainable.\par
%
\begin{figure}
	\centerline{\includegraphics[width=0.75\columnwidth]{mechanism/SI-Fig20.eps}}
	\caption[Comparison of Int-2 formed on- and off-enzyme]{Comparison of Int-2 formed on- and off-enzyme. (Left) EIC traces showing Int-2 produced as a result of spontaneous Int-1 decay in the absence of enzyme (see Figure~\ref{fig:on-or-off-enzyme}). A single Int-2 peak is observed in this case. A small peak for Int-2 was observed in the time-zero fraction. Based on examining the preceding blank, this is likely carry-over from previous runs isolating Int-1. The vertical axis is scaled to a fixed intensity across all samples since only Int-2 is shown in this case. (Right) EIC traces showing Int-2 produced in the presence of enzyme (taken from Figures~\ref{fig:app-TC2-D554N-E10} (1, 2 and 3 minute time points) and~\ref{fig:app-TC3-D554N-E10} (15 and 30 minute time points). These traces are similarly scaled vertically to a fixed intensity since only Int-2 is shown.}
	\label{fig:only-one-AP-without-OGT}
\end{figure}
%
Together with Jacob Sanders, we investigated the feasibility of each possibility computationally. For a model dipeptide (Figure~\ref{fig:imide-amide-model}), he computed the \textit{s}-cis and \textit{s}-trans conformers, as well as the transition state for rotation about the imide bond. These values were then compared to those obtained for the corresponding amide. We used the Eyring–-Polanyi equation (Equation~\ref{Eyring_Polanyi}, where k\textsubscript{B} is Boltzmann's constant, R is the gas constant, and h is Planck's constant) to relate rate and the computed free-energy values. The barrier to rotation for the amide was 22.5 kcal/mol (at 298 K), consistent with values previously reported~\cite{Kern:1997by,Scherer:1998it}. In the case of the imide, the rotational barrier was 12.8 kcal/mol (at 298 K), which is more in-line with those seen for esters. These free-energy differences correspond to rates of \textit{s}-cis $\rightarrow$ \textit{s}-trans interconversion of $\approx 2.0 \times10^{-4}$ s$^{-1}$ and $\approx 2.6 \times10^{4}$ s$^{-1}$, for the amide and imide models, respectively. This rate of exchange for the imide would be inconsistent with observing two peaks by LC for Int-2.\par
%
\begin{equation} \label{Eyring_Polanyi}
	k = \frac{k_B T}{h}e^{\frac{-\Delta G^{\circ}}{RT}}
\end{equation}
%
\begin{figure}
	\centerline{\includegraphics[width=0.6\columnwidth]{mechanism/imide-amide-model-1.eps}}
	\caption[Model dipeptides for imide vs. amide bond rotation]{Model dipeptides for imide vs. amide bond rotation. Rotation about the bolded bond (colored in red) is examined.}
	\label{fig:imide-amide-model}
\end{figure}
%
%
\begin{figure}
	\centerline{\includegraphics[width=0.8\columnwidth]{mechanism/imide-rotation-1.eps}}
	\caption[Computed structures for \textit{s}-trans $\rightarrow$ \textit{s}-cis interconversion of a model imide]{Computed structures for \textit{s}-trans $\rightarrow$ \textit{s}-cis interconversion of a model imide. Free-energy diagram not to scale.}
	\label{fig:imide-calculated}
\end{figure}
%
%
\begin{figure}
	\centerline{\includegraphics[width=0.8\columnwidth]{mechanism/amide-rotation-1.eps}}
	\caption[Computed structures for \textit{s}-trans $\rightarrow$ \textit{s}-cis interconversion of a model amide]{Computed structures for \textit{s}-trans $\rightarrow$ \textit{s}-cis interconversion of a model amide. Free-energy diagram not to scale.}
	\label{fig:amide-calculated}
\end{figure}
%
At near-neutral pH (cleavage reaction conditions), peptide epimerization is extremely slow. Thus, in order for the two Int-2 species to be diastereomers, a proton in the Int-2a would have to be epimerization-prone. There is precedent for succinimide-containing peptides to be epimerization prone~\cite{Geiger:1987ti}, and it has been previously demonstrated that the C$\alpha$ proton epimerizes on the order of $10^5$ times faster than the corresponding proton in an acyclic structure (Figure~\ref{fig:epimerization-models}a, red proton)~\cite{Radkiewicz1996ab}. We calculated the energy of enolates formed by abstraction of either H\textsubscript{a} or H\textsubscript{a} for a series of model internal pyroglutamate-containing dipeptides (cyclic) (Figure~\ref{fig:epimerization-models}b), and the corresponding glutamate-containing peptides (acyclic). Since we observed two Int-2 species regardless of the identity of the residue at position 9, we computed both cysteine and alanine dipeptides.\par
%
\begin{figure}
	\centerline{\includegraphics[width=0.9\columnwidth]{mechanism/epimerization-models.eps}}
	\caption[Model systems to probe epimerization of an internal pyroglutamate]{Model systems to probe epimerization of an internal pyroglutamate.}
	\label{fig:epimerization-models}
\end{figure}
%
There was little difference between the calculated pK\textsubscript{a} values for proton leading to ``Enolate B'' in the context of glutamate- or pyroglutamate-containing model dipeptides (Figures~\ref{fig:alanine-epimerization}, \ref{fig:thiolate-epimerization} and \ref{fig:thiol-epimerization}). Strikingly, the pK\textsubscript{a} for the proton leading to ``Enolate A'' was on the order of 10 pK\textsubscript{a} units \emph{lower} for peptides with a pyroglutamate residue (cyclic) (Figures~\ref{fig:alanine-epimerization} and \ref{fig:thiolate-epimerization}). Results are summarized in Table~\ref{tab:pka-summary}. ``Enolate A'' could not be computed for the cysteine thiol-derived peptide (X = SH) as this species underwent an E1cb-type elimination of a hydrogen sulfide anion. These calculations suggest that Int-2 may be prone to epimerization at the C$\alpha$--H of Cys9.\par
%
If Int-2b is indeed the diastereomer of Int-2a, conducting the cleavage reaction in D\textsubscript{2}O should lead to deuterium incorporation at the C$\alpha$ position of Cys9. After amide bond hydrolysis occurs this position will no longer be exchangeable, making isotope incorporation stable. Experiments to test this hypothesis are underway.\par
%
%
\begin{table}
    \begin{center}
        \begin{tabular}{c c c}
        \hline
         & \multicolumn{2}{c}{pK\textsubscript{a}}\\ \hline
        Structure & Glu & pyroGlu\\
        \hline
        Cysteine thiol & & \\ 
        enolate A & N.D.$\ddagger$ & 13.8 \\ 
        enolate B & 26.5 & 27.1 \\
        \hline
        Cysteine thiolate & & \\ 
        enolate A & 29.5 & 21.1 \\ 
        enolate B & 28.0 & 28.0 \\ 
        \hline 
        Alanine & & \\ 
        enolate A & 26.2 & 15.7 \\ 
        enolate B & 26.9 & 26.5 \\
        \hline
        \end{tabular}
        \caption[Summary of calculated pK\textsubscript{a}s]{Summary of calculated pK\textsubscript{a}s. pK\textsubscript{a} values for model dipeptides shown in Figures~\ref{fig:alanine-epimerization}, \ref{fig:thiolate-epimerization} and \ref{fig:thiol-epimerization}. $\ddagger$~E1cb elimination observed.}
        \label{tab:pka-summary}
    \end{center}
\end{table}
%
\begin{figure}[ht]
	\centerline{\includegraphics[width=0.9\columnwidth]{mechanism/enolates-alanine.eps}}
	\caption[Computed structures of Ala-Glu and Ala-pyroGlu model enolates]{Computed structures of Ala-Glu and Ala-pyroGlu model enolates.}
	\label{fig:alanine-epimerization}
\end{figure}
%
%
\begin{figure}[ht]
	\centerline{\includegraphics[width=0.9\columnwidth]{mechanism/enolates-thiolate.eps}}
	\caption[Computed structures of Cys-Glu and Cys-pyroGlu model enolates (thiolate form)]{Computed structures of Cys-Glu and Cys-pyroGlu model enolates (thiolate form).}
	\label{fig:thiolate-epimerization}
\end{figure}
%
%
\begin{figure}[ht]
	\centerline{\includegraphics[width=0.9\columnwidth]{mechanism/enolates-thiol.eps}}
	\caption[Computed structures of Cys-Glu and Cys-pyroGlu model enolates (thiol form)]{Computed structures of Cys-Glu and Cys-pyroGlu model enolates (thiol form). ``E1cb'' denotes that the structure underwent an E1cb elimination, precluding the calculation of this species.}
	\label{fig:thiol-epimerization}
\end{figure}
%
\section{Conclusions} %DONE
%
Though these studies provide a mechanistic picture for how OGT effects HCF-1 cleavage, several mechanistic details still require further study. Though mutagenesis data has provided insight into the requirements, or lack thereof, for the residues adjacent to the glutamic acid, their precise roles are still unknown. Furthermore, there is an absence of information regarding the proton-transfer dynamics occurring during the cleavage reaction: what is the source of the proton that is transfered to the GlcNAc upon formation of the pyroglutamate? How is the proton removed from the amide N--H that attacks the glycosyl ester? The mechanistic picture provided from this work currently implicates no residues in OGT for catalyzing any part of HCF-1 cleavage beyond the initial glutamate glycosylation. Regarding the internal pyroglutamate species, whether the two species are indeed diastereomers, or whether the fact that there are two species has any biological relevance has yet to be shown explicitly.\par
%
%
Overall, the work reported here illuminates how the glycosyltransferase OGT catalyzes amide bond cleavage. Although the rates of cleavage of the model peptide studied here may appear slow, they are consistent with the slow rates estimated from pulse--chase data for cleavage of HCF-1 in cells \cite{Wilson:1995ec}. The relevant timescale for HCF-1 cleavage is the length of the mammalian cell cycle, and the rates of peptide cleavage measured here are fully compatible with that timescale. To the best of our knowledge, HCF-1 is the first example of a protein that undergoes a physiologically relevant backbone cleavage reaction triggered by glutamate glycosylation, but a glycosidase was recently reported to undergo \textit{in vitro} cleavage and a similar mechanism was proposed \cite{Kotzler:2016br}. Therefore glutamate glycosylation may be a general way to effect amide bond hydrolysis in proteins. OGT may have other cleavage substrates, and because 1-–2\% of genomic space throughout all kingdoms of life is occupied by glycosyltransferases \cite{Varki2009ab,Davies:2005ea}, there may exist other enzymes that catalyze glycosylation-mediated amide bond cleavage.\par
%
\section*{Acknowledgments}
%
I am grateful for support from the National Science and Engineering Research Council (NSERC) of Canada as a PGS-M and PGS-D3 fellowship recipient. This work was funding by a National Institutes of Health grant (R01 GM094263) to Suzanne Walker. I thank Bruker Daltonics (Billerica, MA), specifically Christopher Thompson, who graciously provided access to and assistance with a SolariX XR 7T q-Q-FT-ICR mass spectrometer. This was crucial for obtaining structural information about the glycopeptide intermediate. I would like to thank David J.Vocadlo (Simon Fraser University, Vancouver, BC, Canada) for providing UDP-5\textit{S}GlcNAc. I also thank JoAnne Stubbe (MIT), Daniel Kahne, and Walker lab members Zebulon G. Levine and Sara E. S. Martin for critical reading of the \textit{Nat. Chem. Biol.} manuscript. B. McKay Wood provided assistance with the q-ToF mass spectrometer in the Walker lab.\par
%
X-ray diffraction data were collected at the National Synchrotron Light Source at Brookhaven National Laboratory (Beamline X25). Coordinates and structure factors were deposited under accession code 5HGV in the PDB.\par
%
\newpage
%
\section{Methods} \label{sec:mechanism-methods}
%
\subsection{Reagents and General Methods}
%
\textsuperscript{14}C-UDP-GlcNAc was obtained from American Radiochemicals (ARC). UDP-GlcNAc was obtained from Promega as a 100 mM solution in water. THP was obtained from Novagen as a 0.5 M solution in water. Calf alkaline phosphatase (CIP) was obtained from New England Biolabs. Peptides were obtained from Biomatik (Cambridge, ON, Canada); their purity was evaluated by HPLC and mass spectrometry, and they were used as received. Lyophilized peptides were dissolved in water to $\approx$5 mM and added to reactions according to the final concentration desired. Peptide concentrations were determined by measuring A\textsubscript{274} ($\lambda$\textsubscript{max}, tyrosine) in water, assuming an extinction coefficient of 1400 M\textsuperscript{–-1}$\cdot$cm\textsuperscript{–-1}. A complete list of peptides used in this study is provided in Table \ref{tab:listofpeptides}. The general cleavage peptide, E10-32, is derived from human HCF-1 repeat 2, but contains Gln13 rather than Glu13. This E13Q point mutant shows no appreciable difference in reaction rate from a peptide bearing the native sequence, and was used so that a single system could be employed for both reaction monitoring and isotope labeling studies. Numbering starts from the second residue in the HCF-1 peptides (valine) to be consistent with HCF-1 repeat numbering in the literature.\par
%
\subsection{Production of HCF-3R constructs}
%
HCF-3R constructs (3R-EAA, 3R-SAA, 3R-DAA and 3R-AAA; see Table~\ref{tab:listhcf3re10}) were expressed and purified as outlined in section~\ref{sec:hcf3r-expression}.\par
%
\subsection{Production of OGT} \label{sec:ogt-purification-2}
%
hOGT4.5 (D554N), used for crystallography, was prepared by Michael B. Lazarus, as previously reported~\cite{Lazarus:2011ip}. ncOGT constructs: wild-type OGT, OGT (D554N), OGT (K842M) (sequences outlined in Table~\ref{tab:ogtconstructs}) were expressed and purified as outlined in section~\ref{sec:ogt-purification-1}.\par
%
\subsection{\textit{In vitro} HCF-3R cleavage for SDS-PAGE analysis}
%
In a 20 $\mu$L reaction volume was mixed: 1 mM UDP-GlcNAc, 1 mM THP, 100 $\mu$M HCF-3R construct, and 0.1 U CIP in a buffer of 20 mM MgCl\textsubscript{2}, 20 mM Tris and 150 mM NaCl at pH 7.4. Either OGT (to a final concentration of 2.5 $\mu$M) or water were added, and reactions were mixed by pipetting then incubated at 37 $^{\circ}$C in a thermomixer to ensure uniform heating. After 6 hours, reactions were quenched by the addition of an equal volume (20 $\mu$L) of 2$\times$Laemmli loading buffer (with $\beta$-mercaptoethanol). Samples were boiled for 10 minutes, and pelleted at 15,000$\times$g for 10 minutes. 5 $\mu$L of sample was loaded per lane onto a 4–-20\% Tris-Gly gradient gel and run for 45 minutes at 200 V. The gel was stained with Coomassie Brilliant Blue overnight, and destained the following day until bands were clearly visible and a suitable background was obtained.\par
%
\subsection{\textit{In vitro} HCF-3R glycosylation for SDS-PAGE (autoradiography) analysis}
%
In a 10 $\mu$L reaction volume was mixed: 1 mM UDP-GlcNAc (100 $\mu$M hot; 900 $\mu$M cold), 1 mM THP, 55 $\mu$M HCF-3R construct, and 0.1 U CIP in a buffer of 20 mM MgCl\textsubscript{2}, 20 mM Tris and 150 mM NaCl at pH 7.4. Either OGT (to a final concentration of 2.5 $\mu$M) or water were added, and reactions were mixed by pipetting then incubated at 37 $^{\circ}$C in a thermomixer to ensure uniform heating. After 5 hours, reactions were quenched by addition of an equal volume (10 $\mu$L) of 2$\times$Laemmli loading buffer (with $\beta$-mercaptoethanol). Samples were boiled for 10 minutes, and pelleted at 15,000$\times$g. 7 $\mu$L of sample was loaded per lane onto a 4-–20\% Tris-Gly gradient gel and run for 5 minutes at 90 V, followed by 65 minutes at 150 V. The gel was dried, using a gel drier and placed in an exposure cassette. A storage phosphor was placed on top and the phosphor and exposed for about 40 hours before being imaged with a Typhoon imager.\par
%
\subsection{\textit{In vitro} HCF-1 cleavage for LC-MS analysis}\label{method-lc-cleavage}
%
Typical cleavage reactions were conducted as follows: 20 $\mu$L reaction volume with components having the following final concentrations: 1 mM UDP-GlcNAc, 1 mM THP, 12 $\mu$M HCF-1 peptide, 0.1 U CIP and 12 $\mu$M OGT in a buffer of 20 mM MgCl\textsubscript{2}, 20 mM Tris and 150 mM NaCl at pH 7.4. OGT was added last and reactions were mixed by pipetting then incubated at 37 $^{\circ}$C in a thermomixer to ensure uniform heating. After the desired reaction time, reactions were quenched by addition of four volumes (80 $\mu$L) of ice-cold methanol supplemented with 0.1\% formic acid. Reactions were then centrifuged at 16,100$\times$g for 10 minutes to remove any precipitated protein (4 $^{\circ}$C), and transferred to fresh tubes. Samples were concentrated to dryness using a speedvac (with T = 30 $^{\circ}$C), for about 60 minutes. Samples were re-suspended in 100 $\mu$L 0.1\% formic acid, mixed by vortexing, and centrifuged for 10 minutes at 15,000$\times$g (4 $^{\circ}$C) to remove any insoluble material. Samples were then transferred to LC vials, and maintained at 4 $^{\circ}$C until they were analyzed.\par
%
\subsection{Enzyme Stoichiometry for LC-MS experiments}
%
Typical reactions, as described in \ref{method-lc-cleavage}, were conducted with a 1:1 peptide:OGT stoichiometry. Where noted, single turnover (STO) experiments were conducted with 3$\mu$M peptide and 27.5 $\mu$M OGT to ensure that all peptide was bound to OGT.\par
%
\subsection{General LC-MS Methods}
%
General measurements of peptides were performed on a q-Exactive Plus FT-mass spectrometer (Thermo Scientific\textsuperscript{TM}) equipped with an Ultimate 3000 uHPLC. Reaction mixture components were separated using a 33 minute reverse phase HPLC method (Table \ref{tab:gradmethod1}); the last half of the LC method was empirically optimized to wash the column and re-equilibrate between runs to maintain column performance. with a Kinetex\textsuperscript{\textregistered} C18 (2.6 $\mu$m particle size, 100 \AA  ~pore size, 2.1 mm ID, 150 mm length) at a constant flow rate of 0.200 mL/minute and column temperature of 35 $^{\circ}$C.  5 $\mu$L of sample was injected, unless otherwise specified. Detection on the q-Exactive Plus mass spectrometer was performed between 300–-2000 \textit{m/z}, using an acquisition target of 3$\times$10$^{6}$, maximum IT of 200 ms at a resolution of 70,000 for MS and 35,000 for MS/MS data. For MS/MS analysis, dynamic exclusion was turned off and a global inclusion list was used to maximize S/N for the targeted analysis. A collision energy of 30 eV and an isolation window of 6 \textit{m/z} was used for higher-energy collision-induced dissociation (HCD).\par
%
For some experiments additional MS/MS information was obtained using a q-ToF (CID). These measurements were conducted on a Bruker Impact HD q-ToF mass spectrometer coupled to an Agilent 1290 uHPLC. A collision energy of 35 eV and an isolation window of 6 \textit{m/z} was used for collision-induced dissociation (CID). The source and ToF parameters were optimized for the transmission of higher molecular weight ions. A counter current drying gas flow rate of 12 L/min., and a nebulizing gas pressure of 45 bar were used. Funnel 1 was set to 150 V, and funnel 2 was set to 36 V, with a hexapole RF of 300 V\textsubscript{p--p}. A calibration segment was present at the beginning and end of the run for \textit{m/z} calibration with sodium formate cluster using the Bruker HPC algorithm for the curve fitting.\par
%
Unless otherwise stated, extracted ion chromatograms displayed are for the indicated \textit{m/z} $\pm5$ ppm tolerance.\par
%
\subsection{Purification of glycopeptide intermediate by LC} \label{gp-purification}
%
In order to obtain a suitable quantity of glycopeptide intermediate, four parallel reactions were run as follows: 20 $\mu$L reaction volume with components having the following final concentrations: 1 mM UDP-GlcNAc, 1 mM THP, 12 $\mu$M E10-32, 0.1 U CIP and 12 $\mu$M OGT (D554N) in a buffer of 20 mM MgCl\textsubscript{2}, 20 mM Tris and 150 mM NaCl at pH 7.4. OGT was added last and reactions were mixed by pipetting then incubated at 37 $^{\circ}$C in a thermomixer to ensure uniform heating. After 40 seconds, reactions were quenched by addition of four volumes (80 $\mu$L) of ice-cold methanol supplemented with 0.1\% formic acid. Reactions were then centrifuged at 16,100$\times$g for 10 minutes to remove any precipitated protein (4 $^{\circ}$C), and transferred to fresh tubes. Samples were concentrated to dryness using a speedvac (T = 30 $^{\circ}$C) for about 30 minutes. Samples were combined by first re-suspending one sample in 150 $\mu$L 0.1\% formic acid, vortexing, and using the solution to re-suspend the next sample, etc. The final sample was centrifuged for 10 minutes at 15,000$\times$g (4 $^{\circ}$C) to remove any insoluble material. The sample was then transferred to an LC vial and maintained at 4 $^{\circ}$C. In order to simultaneously detect and collect the glycopeptide, I employed an in-line ‘T’ between the column and the mass spectrometer inlet. To compensate for this change, the flow rate was adjusted to 0.2 mL/min, and the instrument tuning was adjusted for maximum sensitivity under these conditions. For each run, 10 $\mu$L of sample was injected; each sample collected was concentrated at 30 $^{\circ}$C by speedvac and when completely dry these were kept on ice until all had been collected. Collected fractions were combined when possible and dried by the same method until the entire initial sample had been purified. This sample was stored dry at –-80 $^{\circ}$C until analysis was performed. An aliquot of one collected fraction was re-injected (1:5 dilution) to evaluate purity and stability, and showed glycopeptide to be the predominant peak in the sample, with some starting material (which happens to elute at a close retention time) as a contaminant. Attempts to exclude starting material from the sample resulted in a low recovery of glycopeptide intermediate, likely due to the low volumes being collected. I cannot exclude the possibility that some hydrolysis of this glutamyl ester contributes to the starting material observed in the purified material.\par
%
\subsection{Reaction of purified glycopeptide intermediate}
%
Purified glycopeptide intermediate, obtained as described in \ref{gp-purification}, was resuspended in neutral water. Equal volumes of glycopeptide were distributed into each of seven reaction tubes. In a 20 $\mu$L final reaction volume, glycopeptide was added to buffer containing 1 mM THP, 20 mM MgCl\textsubscript{2}, 20 mM Tris and 150 mM NaCl at pH 7.4. At specified time points, the reactions were quenched by the addition of four volumes (80 $\mu$L) of ice-cold methanol supplemented with 0.1\% formic acid. Samples were treated as described for standard LC-MS experiments, except dried samples were reconstituted in 20 $\mu$L rather than 100 $\mu$L.\par
%
\subsection{Electron-capture dissociation (ECD) MS/MS experiments}
%
A Bruker 7.0 T Solarix Q-q-FT-ICR mass spectrometer was used for electron capture dissociation (ECD) measurements (and ETD measurements). A dry sample of the glycopeptide intermediate was re-suspended in 40 $\mu$L of 50\% acetonitrile/water supplemented with 0.1\% formic acid. The sample was introduced at a rate of 20 $\mu$L/h using a syringe pump and a Captive Spray\textsuperscript{TM} nanospray source. A capillary voltage of 1000 V was used to get a stable nanospray signal. The quadruply charged species, [M+4H]\textsuperscript{4+}, for the glycopeptide was isolated using the quadrupole and subjected to ECD in the ICR cell using an ECD voltage of 1.0 V, and a time of 0.100 s. A two-megabyte acquisition file size was used to acquire 100 scans that were summed to obtain a single ECD mass spectrum. The data was internally calibrated using single point lock-mass correction using the predicted monoistopic \textit{m/z} of 896.9017 for the precursor ion after an external multi-point linear calibration with arginine clusters. Data were manually inspected for the presence or absence of predicted ECD fragment ions (as determined using the UCSF MS-Product server) within a 5 ppm mass accuracy window.\par
%
\subsection{CID and HCD MS/MS experiments}
%
Structural information about the anhydropeptide species were obtained by MS/MS using both q-Exactive (HCD) and q-ToF (CID) instruments. For this species, and given the instrument tuning, these instruments provided complementary information, with the q-ToF datasets including better data for higher molecular weight fragment ions, particularly those with higher charge states. In order to unequivocally interpret the MS/MS data obtained for the anhydropeptide intermediate we compared it to the MS/MS spectrum of the starting material, E10-32, under identical conditions. In the case of both peptides serine/threonine dehydration in the C-terminal region of the peptide was observed as a result of CID fragmentation; only the anhydropeptide species that forms in the reaction showed dehydration in the Cys9--Glu10 region. Data were then manually inspected for predicted fragment ions (as determined using the UCSF MS-Product server) within a 5 ppm mass accuracy window.\par
%
\subsection{Crystallization of hOGT4.5 (D554N)}
Ternary complexes were prepared by incubating hOGT4.5 (D554N) at 7 mg/mL with 1 mM UDP-GlcNAc and 3 mM CKII3K peptide. Crystals of the UDP-hOGT4.5 (D554N)-CKII3K glycopeptide ternary product complex were obtained by seeding the hOGT4.5 (D554N) protein with wild-type crystals, using the seed bead kit (Hampton) after mixing 2 $\mu$L of complex with 1 $\mu$L of reservoir consisting reservoir containing 1.6 M Li\textsubscript{2}SO\textsubscript{4} and 0.1 M Bis Tris Propane, pH 7.0, as previously reported~\cite{Lazarus:2012gw}. Crystals were cryoprotected in solutions consisting of the reservoir solution, supplemented with 28\% xylitol and flash frozen in liquid nitrogen.\par
%
\subsection{Data Collection, Structure Determination, and Refinement}
%
Data were collected at NSLS beamline X25 at Brookhaven National Laboratory. Data was processed using iMosflm~\cite{Battye:ba5160} and scaled with Scala or Aimless in the CCP4 crystallography suite~\cite{Winn:2011kh}. The PHENIX software package was used for molecular replacement and refinement~\cite{Adams:2010fk}. The previously determined OGT-CKII glycopeptide ternary product complex (PDB: 4GYW) was used as a search model for molecular replacement, using the program Phaser. Models were subsequently refined by rigid body refinement and multiple rounds of simulated annealing, minimization, atomic displacement parameter (ADP or B-factor) refinement and TLS refinement (determined using the TLSMD server~\cite{Painter:2006gy}), with interspersed manual adjustments using Coot~\cite{Emsley:2010cw}. Geometric restraints for UDP-GlcNAc were generating using PHENIX Elbow, and these restraints were used throughout refinement. All structural figures were made with Pymol~\cite{PyMOL} except for the electrostatic surface figures, which were created with CCP4mg~\cite{McNicholas:2011bd}. Crystallographic software accessed through SBGrid~\cite{Morin:2013bs}.\par
%
\subsection{Computational methods} %for $\alpha$-C--H acidity
%
The dipeptide Cys9-Gln10 was extracted from the small peptide present in PDB 4N3B, and was capped with an acetyl group at the N-terminus and an N-methyl group at the C-terminus based on the positions of the two residues flanking the dipeptide in the crystal structure. Gln10 was mutated \textit{in silico} to glutamate to generate a model of the acyclic dipeptide, and the side chain of Gln10 was cyclized \textit{in silico} onto its backbone amide nitrogen to generate a model of the cyclic dipeptide. For both cyclic and acyclic dipeptides, the structure along with two conjugate base structures deprotonated at the alpha positions of Cys9 and Glu10 were optimized in Gaussian \textsc{\char13}09~\cite{g09} at the B3LYP/6-31G+(d)~\cite{Becke1993,Lee1988} level of theory using an IEFPCM~\cite{Miertus1981,Miertus1982,Pascual-ahuir1994} model for the water solvent; a frequency calculation was used to confirm the presence of local minima and calculate free energies at 298 K. All calculations were repeated with Cys9 present as a thiolate and mutated to alanine.  To estimate pK\textsubscript{a} values from free energy differences between each acid and conjugate base, the related molecule N,N'-dimethylacetamide was used as a standard, with its computed free energy difference calibrated to the known experimental pKa value of 29.4 in water~\cite{Richard:2002co}. Structures were rendered in CYLview~\cite{CYLview}.\par
%
The dipeptide model for amide/imide bond rotation was generated as described above. $\Delta$G$^{\circ}$ values were calculated at 298 K.\par
%
%