Kinetic Analysis of Successive Reactions Catalyzed by Bovine Cytochrome P450 17α,lyase

115

Human cytochrome P450 (P450) 2A6 catalyzes 7-hydroxylation of coumarin, and the reaction rate is enhanced by cytochrome b 5 (b 5 ). 7-Alkoxycoumarins were O-dealkylated and also hydroxylated at the 3-position. Binding of coumarin and 7-hydroxycoumarin to ferric and ferrous P450 2A6 are fast reactions (k on ϳ 10 6 M ؊1 s ؊1 ), and the k off rates range from 5.7 to 36 s ؊1 (at 23°C). Reduction of ferric P450 2A6 is rapid (7.5 s ؊1 ) but only in the presence of coumarin. The reaction of the ferrous P450 2A6 substrate complex with O 2 is rapid (k > 10 6 M ؊1 s ؊1 ), and the putative Fe 2؉ ⅐O 2 complex decayed at a rate of ϳ0.3 s ؊1 at 23°C. Some 7-hydroxycoumarin was formed during the oxidation of the ferrous enzyme under these conditions, and the yield was enhanced by b 5 . Kinetic analyses showed that ϳ 1 ⁄3 of the reduced b 5 was rapidly oxidized in the presence of the Fe 2؉ ⅐O 2 complex, implying some electron transfer. High intrinsic and competitive and non-competitive intermolecular kinetic deuterium isotope effects (values 6 -10) were measured for O-dealkylation of 7-alkoxycoumarins, indicating the effect of C-H bond strength on rates of product formation. These results support a scheme with many rapid reaction steps, including electron transfers, substrate binding and release at multiple stages, and rapid product release even though the substrate is tightly bound in a small active site. The inherent difficulty of chemistry of substrate oxidation and the lack of proclivity toward a linear pathway leading to product formation explain the inefficiency of the enzyme relative to highly efficient bacterial P450s. P4501 enzymes are involved in the oxygenation of a variety of natural products and xenobiotic chemicals in microbial systems (3, 4). Much is known about the structure, function, and catalytic features of some of the P450s, particularly the more extensively studied of the bacterial P450s (4, 5). In mammalian systems P450s oxidize many drugs, steroids, carcinogens, fatty acids and eicosanoids, fat-soluble vitamins, and other endobiotic and xenobiotic chemicals (6). Less information is available about the biochemical details of most of the 57 human P450s (7). In particular, the basis of the inherently lower catalytic activities of these and other mammalian P450s relative to some of the microbial forms is not clear.P450 2A6 is a low-to-medium abundance P450 in human liver (7-9) and is also expressed in some extrahepatic tissues (10). The history of this gene/protein goes back to Phillips et al. (11), who identified a human P450 cDNA as a relative of rat P450 2B1. The 7-hydroxylation of coumarin has long been used as an assay of P450 activity in animal and human liver microsomes (12, 13), and Yamano et al. (14) isolated a P450 2A6 cDNA (then termed 2A3) and first showed that the protein derived from heterologous expression had coumarin 7-hydroxylation activity. Miles et al. (15) also provided similar evidence for this particular sequence being associated with coumarin 7-hydroxylation. Our group purified a pr...

mentioning

confidence: 99%

Kinetic Analysis of Oxidation of Coumarins by Human Cytochrome P450 2A6

Yun

Kim

Calcutt

et al. 2005

115

“…NADPH-cytochrome P450 reductase in the microsomal membranes supplies the first and the second electrons to P450s in the same membrane. Although the supply of the second electron to P450 is often the rate-limiting step in P450 reactions, results of recent experiments have revealed that some P450 reactions have rate-determining steps other than in the electron supply (1)(2)(3)(4)(5). Bell and Guengerich (1) clearly showed in the oxidation of ethanol catalyzed by P450 2E1 1 that the kinetic deuterium isotope effect on K m with no effect on k cat was attributed to the rate-limiting product release.…”

mentioning

confidence: 99%

The Rate-determining Step in P450 C21-catalyzing Reactions in a Membrane-reconstituted System

Kominami

Owaki

Iwanaga

et al. 2001

Self Cite

Adrenal cytochrome P450 C21 in a membrane-reconstituted system catalyzed 21-hydroxylation of 17␣-hydroxyprogesterone at a rate higher than that for progesterone in the steady state at 37°C. The rate of product formation in the steady state increased with the concentration of the complex between P450 C21 and the reductase in the membranes. The complex formation was independent of the volume of the reaction, showing that the effective concentrations of the membrane proteins should be defined with the volume of the lipid phase. The rates of conversion of progesterone and 17␣-hydroxyprogesterone to the product in a single cycle of the P450 C21 reaction were measured with a reaction rapid quenching device. The first-order rate constant for the conversion of progesterone by P450 C21 was 4.3 ؎ 0.7 s ؊1 , and that for 17␣-hydroxyprogesterone was 1.8 ؎ 0.5 s ؊1 at 37°C. It was found from the analysis of kinetic data that the rate-determining step in 21-hydroxylation of progesterone in the steady state was the dissociation of product from P450 C21, whereas the conversion to deoxycortisol was the rate-determining step in the reaction of 17␣-hydroxyprogesterone. The difference in the rate-determining steps in the reactions for the two substrates was clearly demonstrated in the pre-steady-state kinetics.Microsomal cytochrome P450 isozymes are integral membrane proteins responsible for the metabolism of various exogenous and endogenous compounds, including steroid hormones. The monooxygenase reaction of P450 requires two electrons and an oxygen molecule. NADPH-cytochrome P450 reductase in the microsomal membranes supplies the first and the second electrons to P450s in the same membrane. Although the supply of the second electron to P450 is often the rate-limiting step in P450 reactions, results of recent experiments have revealed that some P450 reactions have rate-determining steps other than in the electron supply (1-5). Bell and Guengerich (1) clearly showed in the oxidation of ethanol catalyzed by P450 2E1 1 that the kinetic deuterium isotope effect on K m with no effect on k cat was attributed to the rate-limiting product release. P450 2E1 catalyzes the oxidation of ethanol to acetic acid via acetaldehyde, in which about 90% of the intermediate acetaldehyde is directly converted to acetic acid without dissociation from the active site of the enzyme (2). They showed for the first time in P450 reactions that the reactions for two substrates catalyzed by one molecular species of P450 could have different rate-determining steps (1). We demonstrated in studies on the successive reactions of P450 11␤ and P450 17␣ that the rates of product formation in the steady state were regulated by the rates of product release from the enzymes (3-5). P450 17␣ and P450 11␤ catalyze multistep reactions for the formation of androgens and aldosterone, respectively, where the final products are formed from fractions of the intermediates that do not dissociate from the enzymes (3-5). The same mechanism for diminishing the rate of dissociation of ...

“…9). For bovine P450 17A1, Yamazaki et al (19) used a different approach to calculate corresponding ratios of 0.6 and 5 (cf. 0.1 and 2 in our case, see above), yielding a processivity ratio of ϳ8 (5/0.6).…”

Section: Discussionmentioning

confidence: 99%

“…6, 7, and 9). The relevance of these findings to mammalian P450 17A1 enzymes, which vary in many of their catalytic properties for the ⌬ 4 and ⌬ 5 steroids (19,77), is under investigation.…”

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

Structural and Kinetic Basis of Steroid 17α,20-Lyase Activity in Teleost Fish Cytochrome P450 17A1 and Its Absence in Cytochrome P450 17A2

Pallan

Nagy

Lei

et al. 2015

Background: Fish (and human) P450 17A1 catalyze both steroid 17␣-hydroxylation and 17␣,20-lyase reactions. A second fish P450, 17A2 (51% identical), catalyzes only 17␣-hydroxylation. Results: Crystal structures of zebrafish P450 17A1 and 17A2 and human P450 17A1 are very similar. Conclusion: In kinetic analysis, the two-step oxidation of progesterone is more distributive than for pregnenolone. Significance: Small structural differences are associated with activities of the two fish P450s.