2.7.2.12: acetate kinase (diphosphate)

the mutations decrease kcat in the acetate-forming direction by about 2500-5000fold but have little effect on Km for either substrate compared to the wild type enzyme. This mutant has no detectable activity in the acetyl phosphate-forming direction

DELTAG203/Q323G/M324I

inactive

H117A

mutant is inactive in the direction of acetyl phosphate formation. In the direction of acetate formation, alteration of the His117 results in a 16fold increase in the Km for acetyl phosphate and a 95fold decreased kcat, and for a 1500fold reduction in catalytic efficiency

H172A

mutant is inactive in the direction of acetyl phosphate formation. The His172Ala mutant has a similar Km for acetyl phosphate as wild-type, the kcat value is reduced 970fold

I116A

mutant shows no substantial change in the Km for acetate, mutant has a 26fold decreased kcat. In the direction of acetate formation, the Ile116Ala and Ile116Leu variants both display a mild increase in the Km for acetyl phosphate and decreased kcat, resulting in 22- to 27fold-decreased catalytic efficiencies, respectively

I116L

mutant shows no substantial change in the Km for acetate. In the direction of acetate formation, the Ile116Ala and Ile116Leu variants both display a mild increase in the Km for acetyl phosphate and decreased kcat, resulting in 22- to 27fold-decreased catalytic efficiencies, respectively

Q323A/M324A

the variant displays similar Km values for acetyl phosphate and slightly decreased Km values for phosphate as the wild type enzyme but the kcat value is decreased 19fold resulting in about 11fold reduced catalytic efficiency. In the direction of acetyl phosphate formation, these variant displays slightly increased Km for acetate but no increase in Km for diphosphate and only mild decrease in kcat

Q323G/M324I

the variant displays similar Km values for acetyl phosphate and slightly decreased Km values for phosphate as the wild type enzyme but the kcat value is decreased 8.3fold resulting in about 7fold reduced catalytic efficiency. In the direction of acetyl phosphate formation, these variant displays slightly increased Km for acetate but no increase in Km for diphosphate and only mild decrease in kcat

T223G

mutant shows significant activity in the acetate-forming direction of the assay but does not display activity in the acyl phosphate-forming direction with any acyl substrate

T223P

mutant shows little effect on enzyme activity in the direction of acyl phosphate formation. The enzyme remains capable of utilizing substrates as long as hexanoate, and the Km and kcat values for each substrate are comparable to wild-type. In the direction of acetate formation, the Km for acetyl phosphate increases 4fold, the kcat also shows a 4fold increase, and thus the catalytic efficiency, kcat/Km, is unchanged

V87A

mutant displays 16- to 20fold decreased turnover rates in the direction of acetate synthesis but no change in the Km for acetyl phosphate, and they are inactive in the acetyl phosphate-forming direction

V87G