Active site of triosephosphate isomerase: in vitro mutagenesis and characterization of an altered enzyme.
AUTOR(ES)
Straus, D
RESUMO
We have replaced the glutamic acid-165 at the active site of chicken triosephosphate isomerase with an aspartic acid residue using site-directed mutagenesis. Expression of the mutant protein in a strain of Escherichia coli that lacks the bacterial isomerase results in a complementation phenotype that is intermediate between strains that have no isomerase and strains that produce either the wild-type chicken enzyme or the native E. coli isomerase. The value of kcat for the purified mutant enzyme when glyceraldehyde 3-phosphate is the substrate is 1/1500th that of the wild-type enzyme, and the Km is decreased by a factor of 3.6. With dihydroxyacetone phosphate as substrate, the kcat value is 1/240th that of the wild-type enzyme, and Km is 2 times higher. The value of Ki for a competitive inhibitor, phosphoglycolate, is the same for the mutant and wild-type enzymes, at 2 X 10(-5) M. By treating the enzyme-catalyzed isomerization as a simple three step process and assuming that substrate binding is diffusion limited, it is evident that the mutation of glutamic acid-165 to aspartic acid principally affects the free energy of the transition state(s) for the catalytic reaction itself.
ACESSO AO ARTIGO
http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=397539Documentos Relacionados
- Human triose-phosphate isomerase deficiency: a single amino acid substitution results in a thermolabile enzyme.
- Characterization of Triosephosphate Isomerase Mutants with Reduced Enzyme Activity in Mus Musculus
- Hydroperoxide Isomerase: A New Enzyme of Lipid Metabolism
- In vitro mutagenesis of the herpes simplex virus type 1 DNA polymerase gene results in altered drug sensitivity of the enzyme.
- Altered Proteins with Triosephosphate Isomerase Activity in Suppressor-containing Strains of Bacillus subtilis