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PMID: 1967829 Published · ppublish English Journal Article Research Support, U.S. Gov't, P.H.S.

Searching sequence space by definably random mutagenesis: improving the catalytic potency of an enzyme.

Proceedings of the National Academy of Sciences of the United States of America ·Vol. 87 ·No. 2 ·1990-01-00 ·Pages 696-700

Hermes JD, Blacklow SC, Knowles JR

Abstract

How easy is it to improve the catalytic power of an enzyme? To address this question, the gene encoding a sluggish mutant triose-phosphate isomerase (D-glyceraldehyde-3-phosphate ketol-isomerase, EC 5.3.1.1) has been subjected to random mutagenesis over its whole length by using "spiked" oligonucleotide primers. Transformation of an isomerase-minus strain of Escherichia coli was followed by selection of those colonies harboring an enzyme of higher catalytic potency. Six amino acid changes in the Glu-165----Asp mutant of triosephosphate isomerase improve the specific catalytic activity of this enzyme (from 1.3-fold to 19-fold). The suppressor sites are scattered across the sequence (at positions 10, 96, 97, 167, and 233), but each of them is very close to the active site. These experiments show both that there are relatively few single amino acid changes that increase the catalytic potency of this enzyme and that all of these improvements derive from alterations that are in, or very close to, the active site.

MeSH Terms
Aspartic Acid Carbohydrate Epimerases/genetics Escherichia coli/enzymology,genetics Genes, Bacterial Glutamates Glutamic Acid Kinetics Models, Molecular Mutation Oligonucleotide Probes Plasmids Protein Conformation Triose-Phosphate Isomerase/genetics,metabolism
Chemicals
Glutamates Oligonucleotide Probes Aspartic Acid Glutamic Acid Carbohydrate Epimerases Triose-Phosphate Isomerase
Authors & Affiliations
3 authors, click to expand affiliations / ORCID
Hermes J D
Department of Chemistry, Harvard University, Cambridge, MA 02138.
Blacklow S C
Knowles J R
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Article Info
Journal
Proceedings of the National Academy of Sciences of the United States of America
Abbr.
Proc Natl Acad Sci U S A
ISSN
0027-8424
DOI
10.1073/pnas.87.2.696
Published
1990-01-00
Pages
696-700
Language
English
Region
United States
NLM ID
7505876
PMCID
PMC53332
Subset
IM
External Links
PubMed source DOI: 10.1073/pnas.87.2.696 PMC Full Text View journal details
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