Document Detail


Substrate specificity of natural variants and genetically engineered intermediates of Bacillus lentus alkaline proteases.
MedLine Citation:
PMID:  8796329     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
Three natural variants of subtilisin lentus could be differentiated by their amino acid sequence and their specific activity with low molecular weight peptide substrates of the type sAAPFpNA. The variants had amino acid exchanges in five, respective six positions of their amino acid sequence, four of which are located in the substrate loop of the enzyme (positions 92 - 102). Variants of one type of highly alkaline subtilisin (subtilisin 309) were made by site directed mutagenesis, each containing one of the corresponding amino acid exchanges. These intermediate forms were tested for activity, pH-dependence and substrate specificity. The changes in substrate affinity were relatively small for substrates with different amino acids as P1 residue. The differences in activity on peptide-substrates could be related primarily to a single amino acid substitution in the S4 substrate binding pocket in position 102. With substrate variations in the P3 amino acid residue, changes in k(cat) and K(m) revealed the importance of the charged amino acid exchanged between subtilisin 309 and BLAP. By these experiments an interaction of amino acid position 101 and the P3 residue of the substrate could be demonstrated. The substitution of two differently charged amino acids in the substrate binding region resulted in an unchanged pH-profile of the natural enzyme. With the single exchange intermediates differences in the pH-profile could be found, depending on the substrate tested: a characteristic change was observed with casein as substrate, no such change occurred with hemoglobin.
Authors:
K H Maurer; M Markgraf; D Goddette
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Publication Detail:
Type:  Journal Article    
Journal Detail:
Title:  Advances in experimental medicine and biology     Volume:  379     ISSN:  0065-2598     ISO Abbreviation:  Adv. Exp. Med. Biol.     Publication Date:  1996  
Date Detail:
Created Date:  1997-02-27     Completed Date:  1997-02-27     Revised Date:  2008-11-21    
Medline Journal Info:
Nlm Unique ID:  0121103     Medline TA:  Adv Exp Med Biol     Country:  UNITED STATES    
Other Details:
Languages:  eng     Pagination:  243-56     Citation Subset:  IM    
Affiliation:
COGNIS GmbH, Düsseldorf, Germany.
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MeSH Terms
Descriptor/Qualifier:
Alkalies
Amino Acid Sequence
Bacillus / enzymology*
Base Sequence
Genetic Variation*
Hydrogen-Ion Concentration
Insulin / chemistry
Models, Molecular
Molecular Sequence Data
Mutagenesis, Site-Directed*
Oxidation-Reduction
Protein Engineering*
Substrate Specificity
Subtilisins / genetics*
Chemical
Reg. No./Substance:
0/Alkalies; 11061-68-0/Insulin; EC 3.4.21.-/Subtilisins

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine


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