4.2.1.46 C187A 9.4fold decrease in turnover number for dTDP-glucose compared to wild-type value, 6fold decrease in KM-value for dTDP-glucose compared to wild-type value. 8% of the mutant enzyme contains NADH during steady-state turnover by adding a large excess of dTDPglucose, compared to 45% of the wild-type enzyme 650072 4.2.1.46 C187S 4.1fold decrease in turnover number for dTDP-glucose compared to wild-type value, 4fold decrease in KM-value for dTDP-glucose compared to wild-type value. 5% of the mutant enzyme contains NADH during steady-state turnover by adding a large excess of dTDPglucose, compared to 45% of the wild-type enzyme 650072 4.2.1.46 D135135N/E136Q the turnover number for dTDP-6-fluoro-6-deoxyglucose is 340fold lower than that of the wild-type enzyme, the turnover number for dTDP-glucose is 204fold lower than that of the wild-type enzyme 650028 4.2.1.46 D135A 222fold decrease in turnover number for dTDP-glucose compared to wild-type value, 1.2fold increase in KM-value for dTDP-glucose compared to wild-type value. 30% of the mutant enzyme contains NADH during steady-state turnover by adding a large excess of dTDPglucose, compared to 45% of the wild-type enzyme 650072 4.2.1.46 D135A switch from a concerted to stepwise dehydration mechanism is due to the loss of control over the glucosyl C5–C6 bond rotation in the active site 650136 4.2.1.46 D135A the steady-state rate of conversion of dTDP-6-fluoro-6-deoxyglucose to dTDP-4-keto-6-deoxyglucose is identical to that of wild-type. The turnover number for dTDP-6-fluoro-6-deoxyglucose is 1.5fold lower than that of the wild-type enzyme, the turnover number for dTDP-glucose is 223fold lower than that of the wild-type enzyme 650028 4.2.1.46 D135N 124fold decrease in turnover number for dTDP-glucose compared to wild-type value, 1.3fold increase in KM-value for dTDP-glucose compared to wild-type value. 9% of the mutant enzyme contains NADH during steady-state turnover by adding a large excess of dTDPglucose, compared to 45% of the wild-type enzyme 650072 4.2.1.46 D135N switch from a concerted to stepwise dehydration mechanism is due to the loss of control over the glucosyl C5–C6 bond rotation in the active site 650136 4.2.1.46 D135N the steady-state rate of conversion of dTDP-6-fluoro-6-deoxyglucose to dTDP-4-keto-6-deoxyglucose is identical to that of wild-type. The turnover number for dTDP-6-fluoro-6-deoxyglucose is 1.2fold higher than that of the wild-type enzyme, the turnover number for dTDP-glucose is 124fold lower than that of the wild-type enzyme 650028 4.2.1.46 D135N/E136Q 204fold decrease in turnover number for dTDP-glucose compared to wild-type value, 3.2fold increase in KM-value for dTDP-glucose compared to wild-type value. Less than 0.5% of the mutant enzyme contains NADH during steady-state turnover by adding a large excess of dTDPglucose, compared to 45% of the wild-type enzyme 650072 4.2.1.46 E136A 288fold decrease in turnover number for dTDP-glucose compared to wild-type value, 2fold increase in KM-value for dTDP-glucose compared to wild-type value. Less than 0.5% of the mutant enzyme contains NADH during steady-state turnover by adding a large excess of dTDPglucose, compared to 45% of the wild-type enzyme 650072 4.2.1.46 E136A the turnover number for dTDP-6-fluoro-6-deoxyglucose is 690fold lower than that of the wild-type enzyme, the turnover number for dTDP-glucose is 288fold lower than that of the wild-type enzyme 650028 4.2.1.46 E136Q 67fold decrease in turnover number for dTDP-glucose compared to wild-type value, 1.5fold increase in KM-value for dTDP-glucose compared to wild-type value. 3% of the mutant enzyme contains NADH during steady-state turnover by adding a large excess of dTDPglucose, compared to 45% of the wild-type enzyme 650072 4.2.1.46 E136Q the turnover number for dTDP-6-fluoro-6-deoxyglucose is 69fold lower than that of the wild-type enzyme, the turnover number for dTDP-glucose is 67fold lower than that of the wild-type enzyme 650028 4.2.1.46 E198Q 258fold decrease in turnover number for dTDP-glucose compared to wild-type value, 1.8fold increase in KM-value for dTDP-glucose compared to wild-type value. Less than 0.5% of the mutant enzyme contains NADH during steady-state turnover by adding a large excess of dTDPglucose, compared to 45% of the wild-type enzyme 650072 4.2.1.46 E198Q the turnover number for dTDP-6-fluoro-6-deoxyglucose is 190fold lower than that of the wild-type enzyme, the turnover number for dTDP-glucose is 258fold lower than that of the wild-type enzyme 650028 4.2.1.46 H232A 57.6fold decrease in turnover number for dTDP-glucose compared to wild-type value, 1.8fold decrease in KM-value for dTDP-glucose compared to wild-type value 650072 4.2.1.46 H232N 6.8fold decrease in turnover number for dTDP-glucose compared to wild-type value, 1.2fold decrease in KM-value for dTDP-glucose compared to wild-type value. 3% of the mutant enzyme contains NADH during steady-state turnover by adding a large excess of dTDPglucose, compared to 45% of the wild-type enzyme 650072 4.2.1.46 H232Q 114fold decrease in turnover number for dTDP-glucose compared to wild-type value, 1.3fold increase in KM-value for dTDP-glucose compared to wild-type value. Less than 0.5% of the mutant enzyme contains NADH during steady-state turnover by adding a large excess of dTDPglucose, compared to 45% of the wild-type enzyme 650072 4.2.1.46 K164A 15fold increase in Km-value for dTDP-glucose, 820fold decrease in ratio of turnover number to Km-value for dTDP-glucose, 34fold decrease in turnover-number for dTDP-glucose 650089 4.2.1.46 K164M 8.7fold increase in Km-value for dTDP-glucose, 837fold decrease in ratio of turnover number to Km-value for dTDP-glucose, 96fold decrease in turnover-number for dTDP-glucose 650089 4.2.1.46 K199M 115fold decrease in turnover number for dTDP-glucose compared to wild-type value, 1.3fold increase in KM-value for dTDP-glucose compared to wild-type value. 2% of the mutant enzyme contains NADH during steady-state turnover by adding a large excess of dTDPglucose, compared to 45% of the wild-type enzyme 650072 4.2.1.46 K199M the turnover number for dTDP-6-fluoro-6-deoxyglucose is 260fold lower than that of the wild-type enzyme, the turnover number for dTDP-glucose is 115fold lower than that of the wild-type enzyme 650028 4.2.1.46 K199R 288fold decrease in turnover number for dTDP-glucose compared to wild-type value, 2.2fold increase in KM-value for dTDP-glucose compared to wild-type value. 2% of the mutant enzyme contains NADH during steady-state turnover by adding a large excess of dTDPglucose, compared to 45% of the wild-type enzyme 650072 4.2.1.46 K199R the turnover number for dTDP-6-fluoro-6-deoxyglucose is 680fold lower than that of the wild-type enzyme, the turnover number for dTDP-glucose is 288fold lower than that of the wild-type enzyme 650028 4.2.1.46 N190A 551fold decrease in turnover number for dTDP-glucose compared to wild-type value, 0.92fold increase in KM-value for dTDP-glucose compared to wild-type value. Less than 0.5% of the mutant enzyme contains NADH during steady-state turnover by adding a large excess of dTDPglucose, compared to 45% of the wild-type enzyme 650072 4.2.1.46 N190D 441fold decrease in turnover number for dTDP-glucose compared to wild-type value, fold 4.2increase in KM-value for dTDP-glucose compared to wild-type value. Less than 0.5% of the mutant enzyme contains NADH during steady-state turnover by adding a large excess of dTDPglucose, compared to 45% of the wild-type enzyme 650072 4.2.1.46 N190H 217fold decrease in turnover number for dTDP-glucose compared to wild-type value, 1.2fold increase in KM-value for dTDP-glucose compared to wild-type value 650072 4.2.1.46 T134A 1.2fold increase in Km-value for dTDP-glucose, 283fold decrease in ratio of turnover number to Km-value for dTDP-glucose, 233fold decrease in turnover-number for dTDP-glucose 650089 4.2.1.46 T134S 3.7fold increase in Km-value for dTDP-glucose, 7.5fold decrease in ratio of turnover number to Km-value for dTDP-glucose, 2fold decrease in turnover-number for dTDP-glucose 650089 4.2.1.46 T134V 3.3fold increase in Km-value for dTDP-glucose, 788fold decrease in ratio of turnover number to Km-value for dTDP-glucose, 237fold decrease in turnover-number for dTDP-glucose 650089 4.2.1.46 Y160A 2.8fold increase in Km-value for dTDP-glucose, 683fold decrease in ratio of turnover number to Km-value for dTDP-glucose, 247fold decrease in turnover-number for dTDP-glucose 650089 4.2.1.46 Y160F 1.2fold increase in Km-value for dTDP-glucose, 234fold decrease in ratio of turnover number to Km-value for dTDP-glucose, 190fold decrease in turnover-number for dTDP-glucose 650089 4.2.1.46 Y301F 117fold decrease in turnover number for dTDP-glucose compared to wild-type value, 5.2fold increase in KM-value for dTDP-glucose compared to wild-type value. 39% of the mutant enzyme contains NADH during steady-state turnover by adding a large excess of dTDPglucose, compared to 45% of the wild-type enzyme 650072 4.2.1.46 Y301F the turnover number for dTDP-6-fluoro-6-deoxyglucose is 31fold lower than that of the wild-type enzyme, the turnover number for dTDP-glucose is 117fold lower than that of the wild-type enzyme 650028