title: Searching sequence space by definably random mutagenesis: improving the catalytic potency of an enzyme.
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.
authors.affiliation: Department of Chemistry, Harvard University, Cambridge, MA 02138.
authors.forename: J D
authors.initials: JD
authors.lastname: Hermes
authors.forename: S C
authors.initials: SC
authors.lastname: Blacklow
authors.forename: J R
authors.initials: JR
authors.lastname: Knowles
mesh_terms: D001224:Aspartic Acid; D002238:Carbohydrate Epimerases / Q000235:genetics*; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D005798:Genes, Bacterial; D005971:Glutamates; D018698:Glutamic Acid; D007700:Kinetics; D008958:Models, Molecular; D009154:Mutation; D015345:Oligonucleotide Probes; D010957:Plasmids; D011487:Protein Conformation; D014305:Triose-Phosphate Isomerase / Q000235:genetics* / Q000378:metabolism
chemical_list: D005971:Glutamates; D015345:Oligonucleotide Probes; D001224:Aspartic Acid; D018698:Glutamic Acid; D002238:Carbohydrate Epimerases; D014305:Triose-Phosphate Isomerase