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

Oligonucleotide-directed mutagenesis was employed to producemutants of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH)of Escherichia coli and Bacillus stearothermophilus. Three differentmutant proteins—His¹⁷⁶ — Asn, Cys¹⁴⁹ — Ser,Cys¹⁴⁹ — Gly—were isolated from one or both of theenzymes. The study of the properties of these mutants has shownthat Cys¹⁴⁹ is clearly responsible for the information of acharge-transfer transition, named the Racker band, observedduring the NAD⁺ binding to apoGAPDH. This result excludes asimilarity between the Racker band and the charge-transfer transitionobserved following the alkylation of GAPDH by 3-chloroacetylpyridine-adenine dinucleotide.