PROPOSED STEADY-STATE KINETIC MECHANISM FOR CORYNEBACTERIUM AMMONIAGENES FAD SYNTHETASE PRODUCED BY ESCHERICHIA-COLI

The bifunctional enzyme, FAD synthetase (FS), from Corynebacterium amm oniagenes was overproduced in Escherichia coli and purified, and its s teady-state kinetic properties were investigated. Although FMN is an i ntermediate product in the conversion of riboflavin to FAD, FMN must b e released after formation, and then rebind for adenylylation. It was shown that adenylylation of FMN is reversible; FAD and pyrophosphate c an be converted to FMN and ATP by the enzyme. In contrast, under the c onditions studied, phosphorylation of riboflavin is irreversible. A me thod is described for analysis of two catalytic cycles, occurring on o ne enzyme, which have a substrate and/or product in common. The bindin g order for the phosphorylation cycle of FS was established as ribofla vin(in), ATP(in), ADP(out), and FMN(out). The order for the adenylylat ion cycle was ATP(in), FMN(in), pyrophosphate(out), and FAD(out). A se t of steady-state constants was determined, and without additional opt imization, these constants were sufficient to describe experimental pr ogress curves for conversion of riboflavin to FAD. In independent stud ies, it was demonstrated that FMN binds to apo-FS with a dissociation constant of 6-7 mu M, which is 2 orders of magnitude higher than the K -D value for riboflavin. For the steady-state kinetic analysis, this r epresents reversible binding of FMN(out) in the phosphorylation cycle (cycle I), which effectively inhibits catalysis in the adenylylation c ycle (cycle II).