The behaviour of leucine aminopeptidase towards thionopeptides.
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ABSTRACT:
SUBMITTER: Beattie RE
PROVIDER: S-EPMC1148113 | biostudies-other | 1987 Jul
REPOSITORIES: biostudies-other
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Project description:The inhibitory activity of 14 racemic phosphonic acid analogs of phenylglycine, substituted in aromatic rings, towards porcine aminopeptidase N (pAPN) and barley seed aminopeptidase was determined experimentally. The obtained patterns of the inhibitory activity against the two enzymes were similar. The obtained data served as a basis for studying the binding modes of these inhibitors by pAPN using molecular modeling. It was found that their aminophosphonate fragments were bound in a highly uniform manner and that the difference in their affinities most likely resulted from the mode of substitution of their phenyl rings. The obtained binding modes towards pAPN were compared, with these predicted for bovine lens leucine aminopeptidase (blLAP) and tomato acidic leucine aminopeptidase (tLAPA). The performed studies indicated that the binding manner of the phenylglycine analogs to biLAP and tLAPA are significantly similar and differ slightly from that predicted for pAPN.
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Project description:To tailor leucine aminopeptidase from Streptomyces septatus TH-2 (SSAP) to become a convenient biocatalyst, we are interested in Phe221 of SSAP, which is thought to interact with the side chain of the N-terminal residue of the substrate. By using saturation mutagenesis, the feasibility of altering the performance of SSAP was evaluated. The hydrolytic activities of 19 mutants were investigated using aminoacyl p-nitroanilide (pNA) derivatives as substrates. Replacement of Phe221 resulted in changes in the activities of all the mutants. Three of these mutants, F221G, F221A, and F221S, specifically hydrolyzed L-Phe-pNA, and F221I SSAP exhibited hydrolytic activity with L-Leu-pNA exceeding that of the wild type. Although the hydrolytic activities with peptide substrates decreased, the hydrolytic activities with amide and methyl ester substrates were proportional to the changes in the hydrolytic activities with pNA derivatives. Furthermore, based on a comparative kinetic study, the mechanism underlying the alteration in the preference of SSAP from leucine to phenylalanine is discussed.
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Project description:BACKGROUND: Leucine aminopeptidase (LAP) has been known to be a housekeeping protease, DNA-binding protein and repressor or activator in the operon regulation of virulence-associated genes in several bacterial species. LAP activity was consistently detected in overnight cultures of Burkholderia pseudomallei, the causative agent of melioidosis and this enzyme was partially purified and characterised in this study. The intra- and inter-species nucleotide and deduced amino acid sequence variation of LAP encoding gene (pepA) was determined. A pepA/PCR-RFLP assay was designed to facilitate the identification of major LAP sequence types amongst clinical and environmental isolates of B. pseudomallei. RESULTS: LAP activity was detected in B. pseudomallei culture supernantants by zymographic analysis. Optimum activity was at pH 9 and stable at 50[degree sign]C. Enhanced enzymatic activity was observed in the presence of metallic ions Mg2+, Ca2+, Na+ and K+. LAP activity was inhibited by EDTA, 1,10-phenanthroline, amastatin, Mn2+ and Zn2+. Sequence analysis of the complete nucleotide and deduced amino acid sequences of LAP-encoding (pepA) gene showed close genetic relatedness to B. mallei (similarity 99.7%/99.6%), but not with B. thailandensis (96.4%/96.4%). Eight pepA sequence types were identified by comparison with a 596 bp DNA fragment encompassing central regions of the pepA gene. A pepA/PCR-RFLP was designed to differentiate pepA sequence types. Based on restriction analysis with StuI and HincII enzymes of the amplified pepA gene, clinical and environmental isolates showed different predominant RFLP types. Type I was the most predominant type amongst 71.4% (65/91) of the clinical isolates, while Type II was predominant in 55.6% (5/9) of the environmental isolates. CONCLUSIONS: This study showed that LAP is a secretory product of B. pseudomallei with features similar to LAP of other organisms. Identification of major LAP sequence types of B. pseudomallei was made possible based on RFLP analysis of the pepA gene. The high LAP activity detected in both B. pseudomallei and B. thailandensis, suggests that LAP is probably a housekeeping enzyme rather than a virulence determinant.
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