C O M M U N I C A T I O N S
Subsequent kinetic analysis using 4-nitrobenzenethiol (a) as the
alkyl accepting substrate indicates that keto-AdoMet is a respectable
substrate of TPMT (Table 1); the calculated kcat/Km value (120 M-1
s-1) is approximately 300-fold lower than the value measured with
AdoMet. Competition assays conducted at varied ratios of AdoMet
to keto-AdoMet indicate these substrates bind to the same site on
TPMT (see SI 4.3).
benzylthiols were not previously considered as substrates of TPMT,
this aliphatic thiol was intended as a negative control. This
serendipitous finding highlights the utility of keto-AdoMet to
identify undescribed MTase activities.
A general concern regarding substrate analogues, in particular
those with intrinsically reactive aziridines or sulfoniums, is whether
the analogue modifies and inactivates enzymes.4,5 Using mass
spectrometry, no modification was detected for either TPMT or
AdoHcy nucleosidase, even in the presence of excess keto-AdoMet.
More importantly, the activities of these enzymes were not affected.
An additional advantage of keto-AdoMet as a labeling reagent is
that multiple turnovers were observed with various substrates (e.g.,
at least 14 for 4-nitrobenzenethiol). We further report that the
analogue is stable for over 2 months when stored under mildly
acidic conditions and at either -20 or -80 °C.
In summary, keto-AdoMet, a readily accessible analogue of
AdoMet, expands the toolbox available to interrogate the biochemi-
cal functions of MTases. When coupled with specific MTases, the
analogue may further serve as a site-specific labeling reagent for
bioconjugation and structural diversification.4,5
Acknowledgment. This work was supported by the Herman
Frasch Foundation (541-HF02 to Z.S.Z.), NIH (1R01AI058146 to
Z.S.Z. and 1F31GM073559 to J.F.A.), and the American Heart
Association (09PRE2300071 to T.Z.) We thank the reviewers for their
constructive comments. This is contribution number 943 from the
Barnett Institute.
Supporting Information Available: Full experimental details, syn-
thesis, and characterization of keto-AdoMet. This material is free of charge
Figure 1. Top: HPLC trace of the erythrocyte lysate reaction containing
4-nitrobenzenethiol in the presence (red) and absence (black) of AdoMet.
Bottom: Chromatogram at 650 nm after reactions with (red) and without (black)
keto-AdoMet followed by derivatization with Alexa Fluor 647 hydroxylamine.
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