A mutation at Gly314 of the β subunit of the Escherichia coli pyridine nucleotide transhydrogenase abolishes activity and affects the NADP(H)‐induced conformational change

Escherichia coli RH1 contains a mutation causing complete loss of pyridine nucleotide transhydrogenase activity. A single base change in the chromosomal DNA resulted in the replacement of Gly314 of the β subunit by a Glu residue. The mutant enzyme was partially purified and its trypsin cleavage products examined. The distinct pattern of polypeptides given by proteolysis of the normal transhydrogenase in the presence of NADP(H) was absent when the mutant enzyme was treated with trypsin. However, the β subunit of the mutant enzyme retained its ability to bind to NAD-agarose. Further substitutions were made at Gly314 converting it to Ala, Val or Cys by the use of site-directed mutagenesis. All substitutions for Gly314 abolished the activity completely. The enzyme containing the Gly314Ala mutation was studied in detail and behaved exactly as the enzyme containing the Gly314Glu mutation. It is concluded that the mutation in the β subunit abolished the NADP(H)-induced conformational change in the mutant enzyme. This conformational change, caused by NADP(H) binding, is required to cleave the normal β subunit at Arg265 by trypsin. The genes encoding the pyridine nucleotide transhydrogenase were completely resequenced and several corrections have been made to the previously published sequence [Clarke et al. (1986) Eur. J. Biochem. 158, 647–653].