REACTION VERSUS SUBSITE STEREOSPECIFICITY OF PEPTIDYLGLYCINE ALPHA-MONOOXYGENASE AND PEPTIDYLAMIDOGLYCOLATE LYASE, THE 2 ENZYMES INVOLVED IN PEPTIDE AMIDATION

Carboxyl-terminal amidation, a required post-translational modificatio n for the bioactivation of many neuropeptides, entails sequential enzy matic action by peptidylglycine alpha-monooxygenase (PARS, EC 1.14.17. 3) and peptidylamidoglycolate lyase (PGL, EC 4.3.2.5). The monooxygena se, PARS, first catalyzes conversion of a glycine-extended pro-peptide to the corresponding alpha-hydroxyglycine derivative, and the lyase, PGL, then catalyzes breakdown of this alpha-hydroxyglycine derivative to the amidated peptide plus glyoxylate. We have previously establishe d that PAM and PGL exhibit tandem reaction stereospecificities, with P ARS producing, and PGL being reactive toward, only alpha-hydroxyglycin e derivatives of absolute configuration (S). We now demonstrate that P ARS and PGL exhibit dramatically different subsite stereospecificities toward the residue at the penultimate position (the P-2 residue) in b oth substrates and inhibitors. Incubation of Ac-L-Phe Gly, Ac-L-Phe-L- Phe-Gly, or (S)-O-Ac-mandelyl-Gly with PAM results in complete convers ion of these substrates to the corresponding alpha-hydroxylated produc ts, whereas the corresponding X-D-Phe-Gly compounds undergo conversion s of < 1%. The K-I of Ac-D-Phe-Gly is at least 700-fold higher than th at of Ac-L-Phe-Gly, and the same pattern holds for other substrate ste reoisomers. This S-2 subsite stereospecificity of PAM also holds for c ompetitive inhibitors; thus, the K-I; of 45 mu M for Ac-L-Phe-OCH2CO2H increases to 2,247 mu M for the -D-Phe- enantiomer. In contrast, incu bation of PGL with Ac-L-Phe-alpha-hydroxy-Gly, Ac-D-Phe-alpha-hydroxy- Gly, (S)-O-Ac-mandelyl-alpha-hydroxy-Gly, or (R)-O-Ac-mandelyl-alpha-h ydroxy-Gly results in facile enzymatic conversion of each of these com pounds to their corresponding amide products. The simultaneous express ion of high reaction stereospecificity and low S-2 subsite stereospeci ficity in the course of PGL catalysis was illustrated by a series of e xperiments in which enzymatic conversion of the diastereomers of Ac-L- Phe-alpha-hydroxy-Gly and Ac-o-Phe-alpha-hydroxy-Gly was monitored dir ectly by RPLC. Kinetic parameters were determined for both substrates and potent competitive inhibitors of PGL, and the results confirm that , in sharp contrast to PAM, the configuration of the chiral moiety at the P-2 position has virtually no effect on binding or catalysis. Thes e results illustrate a case where catalytic domains, which must functi on sequentially (and with tandem reaction stereochemistry) in a given metabolic process, nevertheless exhibit sharply contrasting subsite st ereospecificities toward the binding of substrates and inhibitors.