Off-campus UNL users: To download campus access dissertations, please use the following link to log into our proxy server with your NU ID and password. When you are done browsing please remember to return to this page and log out.
Non-UNL users: Please talk to your librarian about requesting this dissertation through interlibrary loan.
SUBSTRATE SPECIFICITY, INHIBITORS, AND CATALYTIC ELEMENTS OF BACILLUS SUBTILIS AMINOPEPTIDASE
Abstract
Bacillus subtilis aminopeptidase (BSAP) is a monomeric zinc metallopeptidase, Mr = 5200, which contains 1 g-atom of Zn('2+) per mole of protein. BSAP was isolated and purified to a high degree of homogeneity from the culture fluid of Bacillus subtilis by a simplified purification procedure. Substrate specificity studies of native BSAP and Co('2+)-activated BSAP toward amino acid amides, dipeptides, and oligopeptides revealed the enzyme to have a high specificity toward substrates which had positively charged amino acid residues in the NH(,2)-terminal position and hydrophobic amino acid residues in the penultimate position. Several competitive inhibitors of BSAP were found, including mercaptans, hydroxamic acid derivatives and boronic acid derivatives. Nitration of BSAP with a 2000-fold molar excess of tetranitromethane at pH 8.5 caused 90% inactivation in the presence of 60 mM 1-butane-boronic acid. From amino acid analyses, 1 to 2 Tyr residues were shown to be protected by the competitive inhibitor. Acetylation of BSAP by 12 mM N-acetylimidazole at pH 7.5 caused an 80% inactivation in the absence of the competitive inhibitor and a 20% inactivation in the presence of the competitive inhibitor. Spectrophotometric measurements were interpreted to indicate that only one Tyr residue was present in the active site of BSAP. Deacetylation of acetylated BSAP by hydroxylamine restored the original activity. Thus the active site tyrosine was concluded to be catalytically functional in the mechanism of hydrolysis. A model for the active site of BSAP was proposed which possessed a negatively charged site for binding the positively charged (alpha)-L-amino acid of the NH(,2)-terminal residue of substrates. A carboxyl group was suggested to be responsible for the negative charge in this binding site. A hydrophobic binding site was proposed for binding to the penultimate amino acid residue of substrates. Catalytically functional groups included a Zn('2+) ion, which was thought to coordinate to the oxygen atom of the carbonyl group of the scissile peptide bond, and a Tyr residue which serves as a proton donor to the nitrogen atom of the scissile peptide bond. A base, presumably a carboxyl group, functions as a donor of a OH('-) to the hydrolyzed bond.
Subject Area
Biochemistry
Recommended Citation
AJABNOOR, MOHAMMED ALI M, "SUBSTRATE SPECIFICITY, INHIBITORS, AND CATALYTIC ELEMENTS OF BACILLUS SUBTILIS AMINOPEPTIDASE" (1981). ETD collection for University of Nebraska-Lincoln. AAI8124505.
https://digitalcommons.unl.edu/dissertations/AAI8124505