Faculty Collaboration Database

Inhibition of the aminopeptidase from Aeromonas proteolytica by aliphatic alcohols. Characterization of the hydrophobic substrate recognition site. Biochemistry 1999 Aug 31;38(35):11433-9

Date

09/02/1999

Pubmed ID

10471294

DOI

10.1021/bi991090r

Scopus ID

2-s2.0-0033621079 (requires institutional sign-in at Scopus site)   27 Citations

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.

Author List

Ustynyuk L, Bennett B, Edwards T, Holz RC

Author

Brian Bennett D.Phil. Professor and Chair in the Physics department at Marquette University

MESH terms used to index this publication - Major topics in bold

1-Propanol
Aeromonas
Alcohols
Aminopeptidases
Bacterial Proteins
Benzyl Alcohol
Binding, Competitive
Butanols
Circular Dichroism
Electron Spin Resonance Spectroscopy
Ethanol
Hydrogen-Ion Concentration
Methanol
Pentanols
Phenols
Spectrometry, Fluorescence
Substrate Specificity