Proposed steady-state kinetic mechanism for Corynebacterium ammoniagenes FAD synthetase produced by Escherichia coli.
The bifunctional enzyme, FAD synthetase (FS), from Corynebacterium ammoniagenes was overproduced in Escherichia coli and purified, and its steady-state kinetic properties were investigated. Although FMN is an intermediate product in the conversion of riboflavin to FAD, FMN must be released after formation, and then rebind for adenylylation. It was shown that adenylylation of FMN is reversible; FAD and pyrophosphate can be converted to FMN and ATP by the enzyme. In contrast, under the conditions studied, phosphorylation of riboflavin is irreversible. A method is described for analysis of two catalytic cycles, occurring on one enzyme, which have a substrate and/or product in common. The binding order for the phosphorylation cycle of FS was established as riboflavin(in), ATP(in), ADP(out), and FMN(out). The order for the adenylylation cycle was ATP(in), FMN(in), pyrophosphate(out), and FAD(out). A set of steady-state constants was determined, and without additional optimization, these constants were sufficient to describe experimental progress curves for conversion of riboflavin to FAD. In independent studies, it was demonstrated that FMN binds to apo-FS with a dissociation constant of 6-7 microM, which is 2 orders of magnitude higher than the KD value for riboflavin. For the steady-state kinetic analysis, this represents reversible binding of FMN(out) in the phosphorylation cycle (cycle I), which effectively inhibits catalysis in the adenylylation cycle (cycle II).
Efimov
I
I
Molecular Biology Division, Department of Veterans Affairs Medical Center, San Francisco, California 94121, USA.
Kuusk
V
V
Zhang
X
X
McIntire
W S
WS
D000255:Adenosine Triphosphate / Q000378:metabolism; D003352:Corynebacterium / Q000201:enzymology* / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D005486:Flavin Mononucleotide / Q000378:metabolism; D007700:Kinetics; D009097:Multienzyme Complexes / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D009713:Nucleotidyltransferases / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000378:metabolism*; D012256:Riboflavin / Q000378:metabolism
D009097:Multienzyme Complexes; D011994:Recombinant Proteins; D005486:Flavin Mononucleotide; D000255:Adenosine Triphosphate; D009713:Nucleotidyltransferases; C020143:FMN adenylyltransferase; D012256:Riboflavin