1312774-10-9Relevant articles and documents
Looking glass mechanism-based inhibition of peptidylglycine α-amidating monooxygenase
Foster, Michael S.,Oldham, Charlie D.,May, Sheldon W.
, p. 283 - 293 (2011)
Carboxyl-terminal amidation, a required post-translational modification for the bioactivation of many peptide hormones, entails sequential enzymatic action by peptidylglycine α-monooxygenase (PAM, EC 1.14.17.3) and peptidylamidoglycolate lyase (PGL, EC 4.3.2.5). We have previously demonstrated that PAM and PGL exhibit strict tandem reaction stereospecificities, with PAM producing exclusively α-hydroxyglycine moieties of absolute configuration (S), and PGL being reactive only toward (S)-α-hydroxyglycines, and we have also shown that PAM exhibits strict P2-subsite stereospecificity toward both peptide substrates and peptidyl competitive inhibitors. Herein, it is reported that the inhibitory stereochemistry of olefinic mechanism-based amidation inhibitors differs from the strict subsite stereospecificity exhibited by PAM toward substrates and reversible competitive inhibitors. Kinetic analyses of mechanism-based irreversible inhibition of PAM by the (S)- and (R)-enantiomers of 5-acetamido-4-oxo-6-phenyl-2-hexenoic acid were carried out using the rigorous progress curve method. The two enantiomers were found to exhibit very similar values of KI and kinact and in both cases kinetic analysis confirmed that irreversible inhibition occurs strictly at the substrate binding site with no ESI complex being formed during the catalytic processing of these irreversible inhibitors. Molecular docking studies were carried out to help rationalize the sharp contrast in the stereospecificity of PAM toward irreversible inhibitors versus substrates and competitive inhibitors. The results revealed that, in contrast to substrates, both docked enantiomers of the olefinic irreversible inhibitors are well-positioned to undergo catalytic processing at the Cu center that gives rise to irreversible inhibition. Taken together, these results provide one of the first clear examples where the stereospecificity of a particular enzyme toward mechanism-based irreversible inhibitors differs from that for substrates and competitive inhibitors.
