Kinetic dissection of the catalytic mechanism of taurine:α- ketoglutarate dioxygenase (TauD) from Escherichia coli

Recent studies on taurine:α-ketoglutarate dioxygenase (TauD) from Escherichia coli have provided evidence for a three-step, minimal kinetic mechanism involving the quaternary TauD-Fe(II)-α-ketoglutarate-taurine complex, the taurine-hydroxylating Fe(IV)-oxo intermediate (J) that forms upon reaction of the quaternary complex with O₂, and a poorly defined, Fe(II)-containing intermediate state that converts in the rate-limiting step back to the quaternary complex [Price, J. C., Barr, E. W., Tirupati, B., Bollinger, J. M., Jr., and Krebs, C. (2003) Biochemistry 42, 7497-7508]. The mapping of this kinetic mechanism onto the consensus chemical mechanism for the Fe(II)- and α-ketoglutarate-dependent engendered several predictions and additional questions that have been experimentally addressed in the present study. The results demonstrate (1) that postulated intermediates between the quaternary complex and J accumulate very little or not at all; (2) that decarboxylation of α-ketoglutarate occurs prior to or concomitantly with formation of J; (3) that the second intermediate state comprises one or more product complex with Mössbauer features that are partially resolved from those of the binary TauD-Fe(II), ternary TauD-Fe(II)-α-ketoglutarate, and quaternary TauD-Fe(II)-α-ketoglutarate-taurine complexes; and (4) that the rate-determining step in the catalytic cycle is release of product(s) prior to the rapid, ordered binding of α-ketoglutarate and then taurine to regenerate the Ch-reactive quaternary complex. The results thus integrate the previously proposed kinetic and chemical mechanisms and indicate which of the postulated intermediates in the latter will be detectable only upon perturbation of the kinetics by changes in reaction conditions (e.g., temperature), protein mutagenesis, the use of substrate analogues, or some combination of these.