Kinetic characterization of the disulfide bond-forming enzyme DsbB.
DsbB is an integral membrane protein responsible for the de novo synthesis of disulfide bonds in Escherichia coli and many other prokaryotes. In the process of transferring electrons from DsbA to a tightly bound ubiquinone cofactor, DsbB undergoes an unusual spectral transition at approximately 510 nm. We have utilized this spectral transition to study the kinetic cycle of DsbB in detail using stopped flow methods. We show that upon mixing of Dsb-B(ox) and DsbA(red), there is a rapid increase in absorbance at 510 nm (giving rise to a purple solution), followed by two slower decay phases. The rate of the initial phase is highly dependent upon DsbA concentration (k(1) approximately 5 x 10(5) M(-1) s(-1)), suggesting this phase reflects the rate of DsbA binding. The rates of the subsequent decay phases are independent of DsbA concentration (k(2) approximately 2 s(-1); k(3) approximately 0.3 s(-1)), indicative of intramolecular reaction steps. Absorbance measurements at 275 nm suggest that k(2) and k(3) are associated with steps of quinone reduction. The rate of DsbA oxidation was found to be the same as the rate of quinone reduction, suggestive of a highly concerted reaction. The concerted nature of the reaction may explain why previous efforts to dissect the reaction mechanism of DsbB by examining individual pairs of cysteines yielded seemingly paradoxical results. Order of mixing experiments showed that the quinone must be pre-bound to DsbB to observe the purple intermediate as well as for efficient quinone reduction. These results are consistent with a kinetic model for DsbB action in which DsbA binding is followed by a rapid disulfide exchange event. This is followed by quinone reduction, which is rate-limiting in the overall reaction cycle.
Tapley
Timothy L
TL
Department of Molecular, Cellular and Developmental Biology, University of Michigan, Ann Arbor Michigan 48109, USA.
Eichner
Timo
T
Gleiter
Stefan
S
Ballou
David P
DP
Bardwell
James C A
JC
D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism; D004579:Electron Transport / Q000502:physiology; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000378:metabolism; D007700:Kinetics; D008565:Membrane Proteins / Q000737:chemistry* / Q000378:metabolism; D008956:Models, Chemical; D019704:Protein Disulfide-Isomerases / Q000737:chemistry* / Q000378:metabolism; D014451:Ubiquinone / Q000737:chemistry / Q000378:metabolism
D001426:Bacterial Proteins; C079503:DsbB protein, Bacteria; D029968:Escherichia coli Proteins; D008565:Membrane Proteins; D014451:Ubiquinone; D019704:Protein Disulfide-Isomerases