On the effects of replacing the carboxylate-binding arginine-171 by hydrophobic tyrosine or tryptophan residues in the L-lactate dehydrogenase from Bacillus stearothermophilus.
For L-lactate dehydrogenases (LDH's), the interaction of the guanidinium group of their Arg 171 residue with the carboxylate group of an alpha-keto acid is of primary importance in orienting the substrate productively at the active site. LDH's such as that of Bacillus stearothermophilus (BSLDH) are of practical importance for the preparation of chiral 2-hydroxy acids used as synthons in asymmetric synthesis but would even be more valuable in this regard if their specificities were broader. With a view to tailoring the specificity of BSLDH toward carbonyl substrates that lack an alpha-carboxyl group such as ketones, site-directed mutagenesis has been applied to replace Arg 171 by the approximately isosteric, but hydrophobic, amino acids Tyr and Trp. The mutant enzymes exhibit remarkably good catalytic activities toward representative alpha-keto acids RCOCOOH, where R = Me, Et, n-Pr, n-Bu, and CH2OH, although for the mutant enzymes the kcat/KM's are lower by approximately 10(3)-10(4)-fold than those for native BSLDH. Surprisingly, the 171----Tyr/Trp enzymes are significantly more active than 171----Lys (Hart et al., 1987a), for which an interaction of a positively charged side chain with substrate COO- is retained. Preparative-scale 171----Trp catalyzed reduction of pyruvate gave optically pure L-lactate, showing that L stereospecificity of such LDH enzymes was unaffected by the loss of Arg 171. The retention of L stereospecificity is attributed to secondary polar or hydrogen-bonding associations of Arg 109 and Thr246, respectively, with the substrate COO-function that are of sufficient magnitude to maintain "normal" substrate orientation.(ABSTRACT TRUNCATED AT 250 WORDS)
Luyten
M A
MA
Department of Chemistry, University of Toronto, Ontario, Canada.
Gold
M
M
Friesen
J D
JD
Jones
J B
JB
D001120:Arginine; D001665:Binding Sites; D001411:Geobacillus stearothermophilus / Q000201:enzymology* / Q000235:genetics; D007700:Kinetics; D007770:L-Lactate Dehydrogenase / Q000235:genetics / Q000378:metabolism*; D015394:Molecular Structure; D009154:Mutation; D013379:Substrate Specificity; D014364:Tryptophan; D014443:Tyrosine
D014443:Tyrosine; D014364:Tryptophan; D001120:Arginine; D007770:L-Lactate Dehydrogenase