Inhibition of the Aminopeptidase from Aeromonas proteolytica by Aliphatic Alcohols. Characterization of the Hydrophobic Substrate Recognition Site^†

Abstract

Seven aliphatic and two aromatic alcohols were tested as reporters of the substrate 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 presence of aliphatic alcohols was addressed. On the basis of circular dichroism (CD), electronic absorption, and fluorescence spectra, the secondary structure 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 increases 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 the 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 absorption 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 inhibition. Combination of these data suggests that each of these inhibitors bind only to the hydrophobic pocket of AAP and, consequently, block the binding of substrate. Thus, the first step in peptide hydrolysis is the recognition of the N-terminal amino acid side chain by the hydrophobic pocket adjacent to the dinuclear active site of AAP.

Potent inhibition of dinuclear zinc(II) peptidase, an aminopeptidase from Aeromonas proteolytica, by 8-quinolinol derivatives: inhibitor design based on Zn2+ fluorophores, kinetic, and X-ray crystallographic study

“…2) with AAP were determined at pH 8.0 [10 mM EPPS with I = 0.1 (NaNO 3 ) containing 2% DMSO] and 25°C ( Table 1). As previously reported [41], the hydrolysis of LeuNA by AAP follows Michaelis-Menten kinetics. The K m value for the hydrolysis of LeuNA at pH 8.0 [10 mM EPPS with I = 0.1 (NaNO 3 ) containing 2% DMSO] determined in this study was 270 ± 10 lM.…”

Heterologous expression and purification of Vibrio proteolyticus (Aeromonas proteolytica) aminopeptidase: A rapid protocol

“…The thermostability of VpAP (see Step C) may be related to this phenomenon. An additional factor that may contribute to the anomalous behavior of unprocessed and partially processed VpAP on SDS-PAGE is the presence of regions of high hydrophobicity in both the mature VpAP sequence [96], and, particularly, in the N-terminal propeptide.…”

Lysine biosynthesis in bacteria: a metallodesuccinylase as a potential antimicrobial target

“…7) [40]. On the basis of the proposed catalytic mechanism for AAP [44, 45], the first step in catalysis for DapE enzymes is likely recognition of the L,L-SDAP side chain by the crescent-shaped cavity adjacent to the Zn1 site. Next, the peptide carbonyl oxygen of L,L-SDAP coordinates to Zn1 and expands its coordination number from four to five, activating the carbonyl for nucleophilic attack.…”