Rate-limiting steps in oxidations catalyzed by rabbit cytochrome P450 1A2.
Several issues regarding the rate-limiting nature of individual reaction steps in catalysis by rabbit liver cytochrome P450 (P450) 1A2 were addressed using anisoles and other substrates. Substrate binding is very fast (k > 10(6) M(-1) s(-1)). Product release is not rate-limiting, as shown by the absence of bursts, placing rate-limiting steps at or before product formation. We had previously shown that the first 1-electron reduction step is fast (k > 700 min(-1)), even in the absence of ligand [Guengerich, F. P., and Johnson, W. W. (1997) Biochemistry 36, 14741-147500]. O(2) binding to ferrous P450 is fast (k >/= 10(6) M(-1) s(-1)). The decay of the P450 Fe(2+)-substrate-O(2) complex was slow in the absence of NADPH-P450 reductase, with a first-order rate constant of 14 min(-1) at 25 degrees C. During the decay, product was formed (from the substrate methacetin) in 61% theoretical yield, although this reaction requires electron transfer among P450 molecules and may not be related to normal turnover. Steady-state spectra suggest that one or more iron-oxygen complexes accumulate, representing entities between the Fe(2+)-O(2) complex and putative FeO(3+) entity. Kinetic isotope effect experiments were done with several substrates, mainly anisoles. Apparent intrinsic deuterium isotope effects as high as 15 were measured. In all cases, the C-H bond-breaking step is at least partially rate-limiting. The isotope effects were not strongly attenuated in noncompetitive or competitive experiments, consistent with relatively rapid P450-substrate exchange, except with the active enzyme Fe-O complex. Kinetic simulations with the available data (i) are consistent with the view that C-H bond breaking is a major rate-limiting step, (ii) demonstrate that increasing the rate of this step will affect k(cat), K(m), and kinetic hydrogen isotope effects but will only increase catalytic efficiency to a certain degree, (iii) indicate that increasing ground-state binding can increase catalytic efficiency but not k(cat), and (iv) suggest that nonproductive binding modes and abortive reduction of O(2) are factors that attenuate catalytic efficiency.
Department of Biochemistry and Center in Molecular Toxicology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146, USA. f.guengerich@vanderbilt.edu
F Peter
FP
Guengerich
Joel A
JA
Krauser
William W
WW
Johnson
D000081:Acetamides / Q000378:metabolism; D000818:Animals; D000840:Anisoles / Q000378:metabolism; D002384:Catalysis; D019388:Cytochrome P-450 CYP1A2 / Q000737:chemistry* / Q000378:metabolism; D003903:Deuterium; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D008956:Models, Chemical; D010084:Oxidation-Reduction; D011485:Protein Binding; D011817:Rabbits
D000081:Acetamides; D000840:Anisoles; C035495:methacetin; D003903:Deuterium; D019388:Cytochrome P-450 CYP1A2