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.
Guengerich
F Peter
FP
Department of Biochemistry and Center in Molecular Toxicology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146, USA. f.guengerich@vanderbilt.edu
Krauser
Joel A
JA
Johnson
William W
WW
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