Steady State and Theoretical Investigations of Peptidylglycine α-Amidating Monooxygenase (PAM)

Approximately 50 percent of all known peptide hormones are post-translationally modified at their C-terminus. These peptide hormones are responsible for cellular functions critical to survival. Peptidylglycine alpha-amidating monooxygenase (PAM) is a bi-functional enzyme which catalyzes the conversion of peptide pro-hormones to peptide hormones. PAM is the only known mammalian enzyme that catalyzes the necessary alpha-amidation to activate these peptide hormones. PAM has previously been found to perform N-dealkylation, as well as O-dealkylation. We report here that a novel chemistry for PAM, S-dealkylation, has now been shown. PAM was able to catalyzes the hydroxylation and subsequent dealkylation for a series of substituted 2- (phenylthio)acetic acid analogs, leaving a product containing a free thiol capable of coordinating to copper(I). A series of cinnamic acid derivatives have been investigated as turnover dependent inactivators of PAM. It was shown that the inactivating compounds contained electron donating substituents. All compounds bound competitively versus substrate, though no catalytic activity was noted when tested as substrates. Although no Dkinact was observed when using perdeuterated cinnamic acid, one cannot rule out hydrogen abstraction from the Cα as this step may not be rate limiting for inactivation. This suggests that the activated oxygen species generated at CuM may be sufficiently reactive to abstract a hydrogen from an alkene to generate a vinyl radical. Substrate activation is believed to be facilitated by a Cu(II)-superoxo complex formed at CuM. Hydrogen abstraction from the Cα is hypothesized to generate a radical, though this has never been demonstrated spectrometrically. We report here further evidence for the generation of an Ca radical by comparing log(Vmax/KO2) vs σ+ for a series of ring-substituted 4-phenyl-3- butenoic acids. Lastly, a computational study was carried out to probe for a possible binding pocket for the reductant, ascorbate. Though crystal structures have argued that reduction of the enzymebound coppers is collisional, kinetic data for inhibitors competitive against ascorbate indicates that a discrete binding pocket may exist. Our study suggests a specific site for binding and provides free energy calculations in agreement with experimental values for binding constants