Dynamic structures of adrenocortical cytochrome P-450 in proteoliposomes and microsomes: protein rotation study

Ohta, Yoshihiro; Kawato, Suguru; Tagashira, Hiroko; Takemori, Shigeki; Kominami, Shiro

doi:10.1021/bi00165a019

Cited by 46 publications

(63 citation statements)

References 48 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Then it was completely degraded at higher proteinase K concentration (1 mg/ml). This result is similar to that obtained from digesting purified bovine P450c21 from lipid vesicles by trypsin (35,36). It indicated that wild type P450c21 was folded into a structure with limited protease entry sites at its surface.…”

Section: Rationale For Site-directed Mutagenesis Of P450c21 At Residusupporting

confidence: 84%

The Common I172N Mutation Causes Conformational Change of Cytochrome P450c21 Revealed by Systematic Mutation, Kinetic, and Structural Studies

Hsu

Guzova

et al. 1996

Journal of Biological Chemistry

View full text Add to dashboard Cite

We have investigated the structure and function of P450c21 with regard to a conserved site around Ile-172 by site-directed mutagenesis making single amino acid substitutions of residues 169 -173. Substitutions of Ile-171 and -172 resulted in production of mutant proteins with dramatic reductions in enzymatic activities, indicating the importance of these two residues in maintaining the structure and function of P450c21. The I171N protein was present at a slightly lower level, due to a decreased rate of protein synthesis. The I172N apoprotein was synthesized at the normal rate, but its hemebound P450 form was present at a much lower level. This I172N protein was tightly integrated into the membrane of endoplasmic reticulum, similar to the wild type P450c21, as shown by immunofluorescence detection, alkaline extraction, and cellular fractionation. Kinetic studies indicated that I172N had a lower V max value. In addition, the I172N protein was more sensitive to proteinase K digestion, indicating a possible alteration of conformation. This conformational change may result in the lower yield of the I172N hemoprotein and the reduced catalytic activity.

show abstract

Section: Rationale For Site-directed Mutagenesis Of P450c21 At Residusupporting

confidence: 84%

The Common I172N Mutation Causes Conformational Change of Cytochrome P450c21 Revealed by Systematic Mutation, Kinetic, and Structural Studies

Hsu

Guzova

et al. 1996

Journal of Biological Chemistry

View full text Add to dashboard Cite

show abstract

“…Kawato and co-workers (16 -20) have extensively studied the interaction between P450 and the reductase in phospholipid vesicles by measuring the rotational diffusion of P450s in the membranes, and they suggested that the mode of interaction depends on the individual P450. Rotational diffusion measurement of P450 C21 in liposomal membranes revealed a complex formation with the reductase (21). In this study, we obtained experimental evidence that the effective concentrations of P450 C21 and NADPH-P450 reductase for the complex formation should be defined with the volume of the lipid phase of the membranes rather than the total volume of the reaction solution.…”

mentioning

confidence: 83%

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

Kominami

Owaki

Iwanaga

et al. 2001

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard 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 ...

show abstract

“…The N-terminal signal anchor sequence is the single membrane-spanning helix in P450s (Sakaguchi et al, 1987;Shimozawa et al, 1993;SzczesnaSkorupa and Kemper, 1993), but the catalytic domain of the protein probably penetrates partially into the membrane (Williams et al, 2000a;Schleinkofer et al, 2005). The orientation and organization of P450s in the membrane are important for their function (Ohta et al, 1992(Ohta et al, , 1994. A second integral membrane protein, cytochrome P450 reductase donates electrons to P450s in their catalytic cycles so both proteins must be oriented properly for optimal electron transfer (Black and Coon, 1982).…”

Section: Introductionmentioning

confidence: 99%

CYP2C8 Exists as a Dimer in Natural Membranes

Johnson

Kemper

2010

Drug Metabolism and Disposition

View full text Add to dashboard Cite

ABSTRACT:CYP2C8 with a modified N-terminal sequence (2C8H) crystallizes as a dimer, but it is not known whether native CYP2C8 exists as a dimer in natural membranes. We have examined the organization of 2C8H and CYP2C8 expressed in bacterial membranes and mammalian endoplasmic reticulum membranes, respectively, by cysteine scanning and cross-linking or oxidation of sulfhydryl groups. In both forms of CYP2C8, cross-linked dimers were observed that were eliminated by mutation of Cys-24 in the linker region. Introduction of individual cysteines in the N-terminal 21-amino acid membrane-spanning signal anchor resulted in a pattern of crosslinking consistent with an ␣-helical structure for the signal anchor. In the linker region, cross-linking was observed for cysteine substituted at residues 22, 23, or 24, just before three Arg residues, indicating close apposition of the two linker sequences despite the neighboring positive charges. Introduction into the F-G loop region of cysteine pairs optimally located for cross-linking based on the crystal structure resulted in cross-linked dimers in the Cys-24 mutant. Deletion of the signal anchor sequence eliminated crosslinking mediated by Cys-24 or by cysteines introduced in the F-G loop regions, indicating that the signal anchor interaction is required for stable dimer formation. These results indicate that the signal anchor sequence and the F-G loop region form interfaces for CYP2C8 intermolecular interactions in natural membranes.

show abstract

Dynamic structures of adrenocortical cytochrome P-450 in proteoliposomes and microsomes: protein rotation study

Cited by 46 publications

References 48 publications

The Common I172N Mutation Causes Conformational Change of Cytochrome P450c21 Revealed by Systematic Mutation, Kinetic, and Structural Studies

The Common I172N Mutation Causes Conformational Change of Cytochrome P450c21 Revealed by Systematic Mutation, Kinetic, and Structural Studies

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

CYP2C8 Exists as a Dimer in Natural Membranes

Contact Info

Product

Resources

About