David J. Nicholls

The importance of arginine 102 for the substrate specificity of Escherichia coli malate dehydrogenase

The malate dehydrogenase from Escherichia coli has been specifically altered at a single amino acid residue by using site-directed mutagenesis. The conserved Arg residue at amino acid position 102 in the putative substrate binding site was replaced with a Gln residue. The result was the loss of the high degree of specificity for oxaloacetate. The difference in relative binding energy for oxaloacetate amounted to about 7 kcal/mol and a difference in specificity between oxaloacetate and pyruvate of 8 orders of magnitude between the wild-type and mutant enzymes. These differences may be explained by the large hydration potential of Arg and the formation of a salt bridge with a carboxylate group of oxaloacetate.

Cloning sequencing and over-expression of Escherichia coli malate dehydrogenase

SummaryThe malate dehydrogenase gene (mdh) from E. coli W3899 has been cloned and its nucleotide sequence determined. A comparison of the DNA sequence presented here and other E. coli mdh gene sequences reveals a number of important differences including an “apparent” frame shift in the C-terminal region of the gene. The mdh gene was shown to be expressed from its own transcription promotor at a very high level (50% soluble cell protein) in this system and a simple single step procedure for purification of the gene product in large quantities is described.

Substitution of the amino acid at position 102 with polar and aromatic residues influences substrate specificity of lactate dehydrogenase

The Gin residue at amino acid position 102 ofBacillus stearothermophilus lactate dehydrogenase was replaced with Ser, Thr, Tyr, or Phe to investigate the effect on substrate recognition. The Q102S and Q102T mutant enzymes were found to have a broader range of substrate specificity (measured bykcat/Km) than the wild-type enzyme. However, it is evident that either Ser or Thr at position 102 are of a size able to accommodate a wide variety of substrates in the active site and substrate specificity appears to rely largely on size discrimination in these mutants. The Q102F and Q102Y mutant enzymes have low catalytic efficiency and do not show this relaxed substrate specificity. However, their activities are restored by the presence of an aromatic substrate. All of the enzymes have a very low catalytic efficiency with branched chain aliphatic substrates.