TABLE 2.
Rates of anaerobic flavin reduction of eNOSr proteins by excess NADPH at 457 nm
Reductions were carried out in the stopped-flow instrument at 10 °C with a 10-fold excess of NADPH while monitoring the absorbance changes at 457 nm. Data were fit to a triple exponential function as described under “Experimental Procedures.”
| Enzyme | Conditions | Dead timea | k1b (%) | k2b (%) | k3b (%) |
|---|---|---|---|---|---|
| % | s−1 | s−1 | s−1 | ||
| Wild type | −CaM | 25 | 24 ± 2 (22) | 1.8 ± 0.1 (18) | 0.9 ± 0.1 (35) |
| +CaM | 27 | 46 ± 4 (16) | 5 ± 0.6 (21) | 0.7 ± 0.1 (36) | |
| S1179D | −CaM | 25 | 44 ± 3 (19) | 5.6 ± 2 (20) | 1.1 ± 0.1 (36) |
| +CaM | 21 | 68 ± 7 (18) | 8 ± 2 (22) | 1.4 ± 0.1 (39) |
a Percentage of the total absorbance change occurring in the instrument dead time.
b Individual rate constants are reported as follows: calculated rate (s−1) (percentage of the total absorbance change for this process).