Use of site-directed mutagenesis to probe the role of Cys149 in the formation of charge-transfer transition in glyceraldehyde-3-phosphate dehydrogenase

Abstract

Oligonucleotide-directed mutagenesis was employed to produce mutants of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) of Escherichia coli and Bacillus stearothermophilus. Three different mutant 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.