The Imino‐Oxy Acetic Acids as Substrates for Peptidylglycine α‐Amidating Monooxygenase (PAM): Product Analysis, Steady‐State Kinetics, and Deuterium Isotope Effects.

Peptidylglycine α‐amidating monooxygenase (PAM) is a bifunctional enzyme consisting of peptidylgycine α‐hydroxylating monooxygenase (PHM) and peptidylglycine amidoglycolate lyase (PAL). The two‐step PAM reaction begins with a stereospecific copper‐ and ascorbate‐dependent hydroxylation of the α‐glycyl carbon in a C‐terminal glycine extended substrate. The subsequent α‐hydroxylated product (PHM) undergoes a zinc‐dependent dealkylation yielding the α‐amidated peptide and glyoxylate (PAL). This reaction is utilized by PAM in the post‐translational bioactivation of ~50% of all known peptide hormones. A variety of novel substrates for PAM that fail to posses a glycine termini, are still able to undergo PAM dependant α‐hydroxylation. Investigation of the imino‐oxy acetic acids (IAA) (PhCX(NOCH 2 C(O)XOH; X=H or CH 2 ) as novel substrates for PAM, shows that PAM oxidized IAAs to their respective oximes and glyoxylate. In contrast to α‐amidated peptide catalysis, product formation appears independent of PAL activity, suggesting that the hydroxylated IAA underwent a rapid, non‐enzymatic dealkylation. The steady state KIE for α‐C‐H cleavage indicates a steady‐state ordered mechanism, with O 2 binding following the imino‐oxy acetic acid, as the D (V MAX /K M ) IAA is strongly dependant on the O 2 concentration and D (V MAX /K M ) oxygen remains constant through variation in IAA concentration.