Triosephosphate isomerase requires a positively charged active site : the role of lysine-12

The role of lysine-12 at the active site of yeast triosephosphate isomerase has been elucidated by a combination of site-directed mutagenesis, Fourier transform infrared spectroscopy, enzyme kinetics, and X-ray crystallography. Several lines of evidence suggest that the mutant isomerase in which lysine has been changed to methionine cannot bind substrate. This mutant enzyme has no detectable catalytic activity, and infrared experiments show no evidence of binding dihydroxyacetone phosphate nor dihydroxyacetone sulfate to the active site. Furthermore, crystals of the enzyme grown in the presence of phosphoglycolohydroxamate, a potent reaction intermediate analog, show an open active site with no inhibitor bound