NDLI: Site-directed mutagenesis and the role of the oxyanion hole in subtilisin.

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Site-directed mutagenesis and the role of the oxyanion hole in subtilisin.

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Site-directed mutagenesis and the role of the oxyanion hole in subtilisin.

Content Provider	World Health Organization (WHO)-Global Index Medicus
Author	Pantoliano, M. W. Hsiao, H. Y. Poulos, T. Bryan, P. Quill, S. G.
Abstract	Oligonucleotide-directed mutagenesis was used to investigate the nature of transition state stabilization in the catalytic mechanism of the serine protease, subtilisin BPN'. The gene for this extracellular enzyme from Bacillus amyloliquefaciens has been cloned and expressed in Bacillus subtilis. In the transition state complex, the carbonyl group of the peptide bond to be hydrolyzed is believed to adopt a tetrahedral configuration rather than the ground-state planar configuration. Crystallographic studies suggest that stabilization of this activated complex is accomplished in part through the donation of a hydrogen bond from the amide side group of Asn-155 to the carbonyl oxygen of the peptide substrate. To specifically test this hypothesis, leucine was introduced at position 155. Leucine is isosteric with asparagine but is incapable of donating a hydrogen bond to the tetrahedral intermediate. The Leu-155 variant was found to have an unaltered Km but a greatly reduced catalytic rate constant, kcat, (factor of 200-300 smaller) when assayed with a peptide substrate. These kinetic results are consistent with the Asn-155 mediating stabilization of the activated complex and lend further experimental support for the transition-state stabilization hypothesis of enzyme catalysis.
ISSN	00278424
e-ISSN	10916490
Journal	Proceedings of the National Academy of Sciences of the United States of America
Issue Number	11
Volume Number	83
Language	English
Publisher	National Academy of Sciences
Publisher Date	1986-07-01
Publisher Place	United States
Access Restriction	Open
Subject Keyword	Genetic Engineering Subtilisins Genetics Amino Acid Sequence Bacillus Enzymology Binding Sites Cloning, Molecular Kinetics Mutation Protein Conformation Structure-Activity Relationship Metabolism Multidisciplinary
Content Type	Text
Resource Type	Article
Subject	Multidisciplinary

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