Role of the histidine 176 residue in glyceraldehyde-3-phosphate dehydrogenase as probed by site-directed mutagenesis.

Laboratoire d'Enzymologie et de Génie Génétique, UA CNRS, 457, Faculté des Sciences, Vandoeuvre les Nancy, France.

The catalytically essential amino acid, histidine 176, in the active site of Escherichia coli glyceraldehyde-3-phosphate dehydrogenase (GAPDH) has been replaced with an asparagine residue by site-directed mutagenesis. The role of histidine 176 as a chemical activator, enhancing the reactivity of the thiol group of cysteine 149, has been demonstrated, with iodoacetamide as a probe. The esterolytic properties of GAPDH, illustrated by the hydrolysis of p-nitrophenyl acetate, have been also studied. The kinetic results favor a role for histidine 176 not only as a chemical activator of cysteine 149 but also as a hydrogen donor facilitating the formation of tetrahedral intermediates. These results support the hypothesis that histidine 176 plays a similar role during the oxidative phosphorylation of glyceraldehyde 3-phosphate.

PMID: 2659073 [PubMed - indexed for MEDLINE]