Active site of triosephosphate isomerase: in vitro mutagenesis and characterization of an altered enzyme.

Abstract

The glutamic acid-165 at the active site of chicken triosephosphate isomerase was replaced 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 .**GRAPHIC**. [catalytic constant] for the purified mutant enzyme when glyceraldehyde 3-phosphate is the substrate is 1/1500 that of the wild-type enzyme, and the .**GRAPHIC**. is decreased by a factor of 3.6. With dihydroxyacetone phosphate as substrate, the .vector..kappa.cat value is 1/240 that of the wild-type enzyme, and .vector.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 .times. 10-5 M. By treating the enzyme-catalyzed isomerization as a simple 3 step process and assuming that substrate binding is diffusion limited, the mutation of glutamic acid-165 to aspartic acid principally affects the free energy of the transition state(s) for the catalytic reaction itself.