Investigation of the proton relay system operative in human cystosolic aminopeptidase P

Abstract

Aminopeptidase P, a metalloprotease, targets Xaa-Proline peptides for cleavage [1–4]. There are two forms of human AMPP, a membrane-bound form (hmAMPP) and a soluble cytosolic form (hcAMPP)[5]. Similar to the angiotensin-I-converting enzyme, AMPP plays an important role in the catabolism of inflammatory and vasoactive peptides, known as kinins. The plasma kinin, bradykinin, was used as the substrate to conduct enzymatic activity analyses and to determine the Michaelis constant (Km) of 174 μM and the catalytic rate constant (kcat) of 10.8 s-1 for hcAMPP. Significant differences were observed in the activities of Y527F and R535A hcAMPP mutants, which displayed a 6-fold and 13.5-fold for decrease in turnover rate, respectively. Guanidine hydrochloride restored the activity of R535A hcAMPP, increasing the kcat/Km 20-fold, yet it had no impact on the activities of the wild-type or Y527F mutant hcAMPPs. Activity restoration by guanidine derivatives followed the order guanidine hydrochloride >> methyl-guanidine > amino-guanidine > N-ethyl-guanidine. Overall, the results indicate the participation of R535 in the hydrogen bond network that forms a proton relay system. The quaternary structure of hcAMPP was determined by using analytical ultracentrifugation (AUC). The results show that alanine replacement of Arg535 destabilizes the hcAMPP dimer and that guanidine hydrochloride restores the native monomer-dimer equilibrium. It is proposed that Arg535 plays an important role in hcAMMP catalysis and in stabilization of the catalytically active dimeric state.

Catalysis of Hydrazone and Oxime Peptide Ligation by Arginine

“…For example, some computational studies suggest that explicit water molecules can promote the dehydration of carbinolamine 6 by facilitating an internal N to O proton shuffling mechanism . Interestingly, arginine is strongly suspected to act as a relay for proton transfers occurring in bacteriorhodopsin, pyridoxal 5′-phosphate synthase, and other proteins. − The question of whether the side-chain guanidinium cation of arginine promotes hydrazone formation by facilitating proton transfers, by acting as a general acid catalyst or by other means will require more investigation.…”