Inhibition of the aminopeptidase from Aeromonas proteolytica by aliphatic alcohols. Characterization of the hydrophobic substrate recognition site

Seven aliphatic and two aromatic alcohols were tested as reporters of the s ubstrate selectivity of the aminopeptidase from Aeromonas proteolytica (AAP ). This series of alcohols was chosen to systematically probe the effect of carbon chain length, steric bulk, and inhibitor shape on the inhibition of AAP. Initially, however, the question of whether AAP is denatured in the p resence of aliphatic alcohols was addressed. On the basis of circular dichr oism (CD), electronic absorption, and fluorescence spectra, the secondary s tructure of AAP, with and without added aliphatic alcohols, was unchanged. These data clearly indicate that AAP is not denatured in aliphatic alcohols , even up to concentrations of 20% (v/v). All of the alcohols studied were competitive inhibitors of AAP with K-i values between 860 and 0.98 mM, The clear trend in the data was that as the carbon chain length increases from one to four, the K-i values increase. Branching of the carbon chains also i ncreases the K-i values, but large bulky groups, such as that found in tert -butyl alcohol, do not inhibit AAP as well as leucine analogues, such as 3- methyl-1-butanol. The competitive nature of the inhibition indicates that t he substrate and each alcohol studied are mutually exclusive due to binding at the same site on the enzyme. On the basis of EPR and electronic absorpt ion data for Co(II)-substituted AAP, none of the alcohols studied binds to the dinuclear metallo-active site of AAP. Thus, reaction of the inhibitory alcohols with the catalytic metal ions cannot constitute the mechanism of i nhibition, Combination of these data suggests that each of these inhibitors bind only to the hydrophobic pocket of AAP and, consequently, block the bi nding of substrate. Thus, the first step in peptide hydrolysis is the recog nition of the N-terminal amino acid side chain by the hydrophobic pocket ad jacent to the dinuclear active site of AAP.