Substitution of the amino acid at position 102 with polar and aromatic residues influences substrate specificity of lactate dehydrogenase.
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.
Division of Biotechnology, Centre for Applied Microbiology and Research, Porton Down, Salisbury, U.K.
D J
DJ
Nicholls
M
M
Davey
S E
SE
Jones
J
J
Miller
J J
JJ
Holbrook
A R
AR
Clarke
M D
MD
Scawen
T
T
Atkinson
C R
CR
Goward
D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D004926:Escherichia coli; D001411:Geobacillus stearothermophilus / Q000201:enzymology*; D005973:Glutamine; D007700:Kinetics; D007770:L-Lactate Dehydrogenase / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D015183:Restriction Mapping; D013379:Substrate Specificity
D011994:Recombinant Proteins; D005973:Glutamine; D007770:L-Lactate Dehydrogenase