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

Abstract

The Gln residue at amino acid position 102 of Bacillus 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 by kcat/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.

Yak Lactate Dehydgogenase A4: Purification, Properties, and cDNA Cloning

“…Third, structure-function analysis of the LDH of the bacterium Bacillus stearothermophilus has elucidated the roles of numerous amino acid residues in governing the enzyme's kinetic properties. [8][9][10][11][12] In this study, we purified and characterized LDH-A4 from skeletal muscle of yak and cattle, and found that yak LDH-A4 Km for pyruvate was much higher than that of cattle. Yak LDH-A cDNA was cloned and analyzed in order to elucidate the amino acid substitutions between yak and cattle.…”

α‐Proteobacterial Relationship of Apicomplexan Lactate and Malate Dehydrogenases

“…The substrate specificity of LDH and MDH is clearly associated with a single amino acid (aa) at the active site loop, in which all MDH enzymes possess a positively charged arginine, whereas most LDH enzymes contain a non‐charged residue. Although site‐directed mutagenesis has shown that the substrate specificity of LDH can easily switch from lactate to malate by a single aa replacement of an uncharged residue to a charged arginine in the active site loop (Boemke et al 1995; Cendrin et al 1993; Nicholls et al 1994; Wilks et al 1988; Wu et al 1999), as yet the reverse has not been demonstrated. Furthermore, there is no evidence that the LDH lineage contains any MDH sequences (Golding and Dean 1998).…”

Evolution of Lactate Dehydrogenase-A Homologs of Barracuda Fishes (Genus Sphyraena) from Different Thermal Environments: Differences in Kinetic Properties and Thermal Stability Are Due to Amino Acid Substitutions Outside the Active Site,

“…First, LDH-A is a slowly evolving protein for which there are extensive data on amino acid sequence (Li et al, 1983;Crawford et al, 1989;Ishiguro et al, 1990;Tsoi & Li, 1994) ( Abad-Zapatero et al, 1987;Gerstein & Chothia, 1991). Second, structure-function analysis of LDH of the bacterium Bacillus stearothermophilus has elucidated the roles of numerous amino acid residues in governing the enzyme's kinetic properties (Wigley et al, 1987(Wigley et al, , 1992Feeney et al, 1990;Deng et al, 1994;Nicholls et al, 1994). Third, the LDH-A homologs of fishes within the genus Sphyraena differ adaptively in kinetic properties (K m of pyruvate and k cat ), although the species have diverged only relatively recently (between approximately 3.5 and 12 million years) and encounter habitat temperatures that differ by only approximately 3-8 °C (Graves & Somero, 1982).…”