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.

No abstract

No abstract

Oligonucleotide-directed mutagenesis was employed to produce mutants of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) of Escherichia coli and Bacillus stearothermophilus. Three different mutants proteins--His176----Asn, Cys149----Ser, Cys149----Gly--were isolated from one or both of the enzymes. The study of the properties of these mutants has shown that Cys149 is clearly responsible for the information of a charge-transfer transition, named the Racker band, observed during the NAD+ binding to apoGAPDH. This result excludes a similarity between the Racker band and the charge-transfer transition observed following the alkylation of GAPDH by 3-chloroacetyl pyridine-adenine dinucleotide.

No abstract