Improved specificity toward substrates with positively charged side chains by site-directed mutagenesis of the L-lactate dehydrogenase of Bacillus stearothermophilus.
The substrate specificities of L-alpha-hydroxy acid dehydrogenases, including L-lactate dehydrogenases (L-LDH's), can often be quite broad. However, an LDH with high catalytic activity toward alpha-keto acids with positively charged side chains, such as those containing ammonium groups, has not been described, even though there is evidence from metabolic studies that a natural dehydrogenase with such activity might exist in Nature. L-omega-Amino-alpha-hydroxy acids are important intermediates in the synthesis of pharmacologically active compounds, and enzymatic reduction of omega-amino-alpha-keto acids represents an attractive route to these compounds. Graphics analysis indicated that introduction of acidic amino acids at position 102 of the L-LDH of Bacillus stearothermophilus (BSLDH) would favor binding of such side chain ammonium groups. Accordingly, Q102E and Q102D mutant BSLDH's were constructed and the steady state kinetic parameters determined for these mutants for a broad range of alpha-keto acids, including omega-amino-keto acids. The results obtained show that, compared to WT-BSLDH, these mutants show up to 25-fold improvements in kcat/Km values for omega-amino-alpha-keto acid substrates.
Hogan
J K
JK
Department of Molecular and Medical Genetics, University of Toronto, Ontario, Canada.
Pittol
C A
CA
Jones
J B
JB
Gold
M
M
D001483:Base Sequence; D002852:Chromatography, Ion Exchange; D004563:Electrochemistry; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D001411:Geobacillus stearothermophilus / Q000201:enzymology*; D007651:Keto Acids / Q000378:metabolism; D007700:Kinetics; D007770:L-Lactate Dehydrogenase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
D007651:Keto Acids; D007770:L-Lactate Dehydrogenase