L-idonate dehydrogenase is an important NAD+/NADH dependent enzyme which catalyzes the oxidation of L-idonate into 5-Ketogluconate in the biosynthesis of tartaric acid from ascorbic acid in grapes. Analysis of the amino acid sequence indicates that L-idonate dehydrogenase from Vitis vinifera is 79% identical to sorbitol dehydrogenase (SDH) from Arabidopsis thaliana. Despite the high degree of homology, each enzyme is specific for its substrate with neither enzyme showing activity towards the other substrate. Mutagenesis of the Wild-type L-IdDH sought to create variants which could bind to sorbitol rather than Idonate. Two variants of the Wild-type L-IdDH were successfully isolated thus allowing for a comprehensive investigation of the enzymatic properties of the two “mutants”. All the derived kinetic constants (Km, Vmax, and Kcat) show that the mutation (Rice) a184g binds one-third as tightly to the substrate 5-ketogluconate while the mutation (Amherst) g395c, t397a binds one-fifth as tightly to 5-Ketogluconate. (Rice) a184g and (Amherst) g395c, t397a catalyze at a rate of one- twentieth slower in comparison to the Wild-type IdDH. The low rate of catalysis is contributed to the change in the amino acid sequence of the mutated L-IdDH cells which leads to poor binding of the enzyme to the substrate.