Science · 1988 · 278 citations · 22 references
Life SciencesBiochemistryMolecular BiologyCatalysisEnzyme MechanismEnzymologyCatalytic MechanismsEnzymatic CatalysisEnzyme CatalysisEnzyme DiscoveryEnzyme KineticsEnzyme-substrate RecognitionCatalytic ActivityDe Novo Enzyme DesignEnzyme Kinetics OptimizationMalate DehydrogenaseMaximum Reaction VelocityMichaelis ConstantSubstrate Binding SpecificitySubstrate Specificity Design
Three variations to the structure of the nicotinamide adenine dinucleotide (NAD)-dependent L-lactate dehydrogenase from Bacillus stearothermophilus were made to try to change the substrate specificity from lactate to malate: Asp197----Asn, Thr246----Gly, and Gln102----Arg). Each modification shifts the specificity from lactate to malate, although only the last (Gln102----Arg) provides an effective and highly specific catalyst for the new substrate. This synthetic enzyme has a ratio of catalytic rate (kcat) to Michaelis constant (Km) for oxaloacetate of 4.2 x 10(6)M-1 s-1, equal to that of native lactate dehydrogenase for its natural substrate, pyruvate, and a maximum velocity (250 s-1), which is double that reported for a natural malate dehydrogenase from B. stearothermophilus.
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Affinities of amino acid side chains for solvent water
Richard Wolfenden, Linda Andersson, Paul M. Cullis et al. · Biochemistry · 1981 · 869 citations