Table 1. Comparison of the Oxidative Half-Reaction Rate Constants of Variants^a.

enzyme	k1 (s–1)	k2 (s–1)	k3 (s–1)b
WT	28.4 ± 0.8	0.20 ± 0.01	–
Q75A	2.2 ± 0.3	0.074 ± 0.002	0.003 ± 0.0005
R90A	0.33 ± 0.01	0.026 ± 0.01	–
R174A	6.8 × 10–5 ± 1.6 × 10–7	–	–
Y91A	0.53 ± 0.04	0.007 ± 0.0001	0.001 ± 0.0002
S88A	0.82 ± 0.05	0.06 ± 0.002	0.019 ± 0.0005

^aAn anaerobic solution of reduced enzyme (14 μM active sites) and dUMP (300 μM) was mixed with 400 μM CH₂THF using a stopped-flow spectrophotometer. The reactions were monitored at 420 nm, and the traces were fit to a sum of exponentials. Reactions were conducted in 0.1 M Tris-HCl (pH 8.0) with 1 mM EDTA and 15 mM CH₂O at 25 °C.

^bSome reaction traces required a third exponential to fit a small increase in absorbance at the end of the reaction. We speculate that this small phase was caused by the reassociation and subsequent oxidation of a small amount of free FAD that dissociated during the preparation.