Reconstituted enzyme systems

DMN oxidative demethylation has been shown to be a liver mi-crosome cytochrome P-450 monooxygenase (10) Lotlikar et al. (11) found that a reconstituted enzyme system, consisting of cytochrome P-450, NADPH-cytochrome P-450 reductase and phosphatidyl choline was effective in catalyzing the demethylation of DMN. The most commonly accepted mechanism for the oxidative demethylation of DMN and, by extension, of other dialkyInltrosamlnes is shown in Scheme 1. 

It was recently reported that. >97% of BaP 4,5-epoxide metabolically formed from the metabolism of BaP in a reconstituted enzyme system containing purified cytochrome P-450c (P-448) is the 4S,5R enantiomer (24). The epoxide was determined by formation, separation and quantification of the diastereomeric trans-addition products of glutathione. Recently a BaP 4,5-epoxide was isolated from a metabolite mixture obtained from the metabolism of BaP by liver microsomes from 3-methylcholanthrene-treated Sprague-Dawley rats in the presence of the epoxide hydrolase inhibitor 3,3,3-trichloropropylene oxide, and was found to contain a 4S,5R/4R,5S enantiomer ratio of 94 6 (Chiu et. al., unpublished results). However, the content of the 4S,5R enantiomer was <60% when liver microsomes from untreated and phenobarbital-treated rats were used as the enzyme sources. Because BaP 4R,5S-epoxide is also hydrated predominantly to 4R,5R-dihydro-... 

An approach for determining the presence of multiple monooxygenases in rat liver microsomes was the use of chemicals that selectively affected certain monooxygenase activities but not others. For example, 7,8-benzoflavone (a-naphthoflavonc) markedly inhibited the hydroxylationofbenzo[a]pyrene by liver microsomes from 3-methylcholanthrene treated rats or by a cytochrome P448-dependent reconstituted enzyme system (68,69), but there was no effect or only a small stimulatory effect of 7,8-benzoflavone on benzo[a]pyrene metabolism in livers from untreated rats (68). 

Purified CYP2C13 exhibits high testosterone hydroxylase activity in a reconstituted enzyme system, but this enzyme makes only marginal contributions to liver microsomal testosterone hydroxylation. ... 

All of our studies with MMO thus far indicate that the regulatory scheme [5] differs from that described for P450 m [14]. In particular, there seems to be no effect of methane and most other substrates on the kinetics of formation of any of the intermediates in the reaction cycle prior to Q [60]. On the other hand, the decay rate of Q depends linearly on the concentration of substrate and leads to product formation, so it is likely that substrate enters the reaction cycle at this stage. This is supported by our recent observation that electron transfer from the reduced MMOR to MMOH occurs readily in the absence of substrate at rates that are not rate limiting for the overall reaction (X.-Y. Zhang and J. D. Lipscomb, unpublished observations). Despite the apparent lack of substrate gating in the MMO catalysis, the reconstituted enzyme system is nearly 100% efiScient in methane turnover. This appears to be due to the effects of component complex formation on the rates of specific steps in the cycle and on the redox potentials of the components [25-27, 60]. 

The 3-ketothiolase has been purified and investigated from several poly(3HB)-synthesizing bacteria including Azotobacter beijerinckii [10], Ral-stonia eutropha [11], Zoogloea ramigera [12], Rhodococcus ruber [13], and Methylobacterium rhodesianum [14]. In R. eutropha the 3-ketothiolase occurs in two different forms, called A and B, which have different substrate specificities [11,15]. In the thiolytic reaction, enzyme A is only active with C4 and C5 3-ketoacyl-CoA whereas the substrate spectrum of enzyme B is much broader, since it is active with C4 to C10 substrates [11]. Enzyme A seems to be the main biosynthetic enzyme acting in the poly(3HB) synthesis pathway, while enzyme B should rather have a catabolic function in fatty-acid metabolism. However, in vitro studies with reconstituted purified enzyme systems have demonstrated that enzyme B can also contribute to poly(3HB) synthesis [15]. 

Fig. 4.1). Since the P450s are membrane-bound enzymes in vivo, adding lipid to reconstitute a functional enzyme system in vitro more closely mirrors the in vivo environment and presumably serves as a matrix to allow the two enzymes, P450 and P450 reductase, to interact properly. 

Yang M, Wang L, Xie G, et al. 1993. Effects of intermediate metabolites of 37 xenobiotics on the catalytic activities of reconstituted cytochrome P-45011B1 and P-4501A1 enzyme systems. Biomed Environ Sci 6 8-26. 

Figure 4. Linearity of the metabolism of parathion and benzphetamine by a reconstituted monooxygenase oxidase enzyme system from rabbit liver. The 0.5-mL reaction mixture contained 50 fig of sodium deoxycholate, 15 iig of dilauroyl l-5-phosphatidylcholine, 1.5 units of NADPH-Cytochrome c reductase, 0.5 nmol of Cytochrome P-450, 0.05M Hepes buffer (pH 7.8), 0.015M MgCh, O.lmU EDTA, and 5 X lO M [ethyl- C] parathion or / X 10 M benzphetamine.

West. S.B. and Lu, A.Y. (1972) Reconstituted liver microsomal enzyme system that hydroxylates drugs, other foreign compounds and endogenous substrates. V. Competition between cytochromes P-450 and P-448 for reductase in 3,4-benzpyrene hydro-xylation. Archives of Biochemistry and Biophysics, 153, 298-303. 

In the catalytic cycle of CYP, reducing equivalents are transferred from NADPH to CYP by a flavoprotein enzyme known as NADPH-cytochrome P450 reductase. The evidence that this enzyme is involved in CYP monooxygenations was originally derived from the observation that cytochrome c, which can function as an artificial electron acceptor for the enzyme, is an inhibitor of such oxidations. This reductase is an essential component in CYP-catalyzed enzyme systems reconstituted from purified components. Moreover antibodies prepared from purified reductase are inhibitors of microsomal... 

Preparations containing a single P450 isozyme are available as either expression systems or purified, reconstituted enzymes. The P450s have been expressed in bacterial, yeast, insect, and mammalian cells (8). Most of these enzymes can be used in the membranes in which they are expressed. However, in order to obtain adequate enzyme activity for most expression systems, it is necessary to supplement the membranes with reductase and in some cases cytochrome b5. This is accomplished by either supplementing the membranes with purified coenzymes or by coexpression of the coenzymes. Alternatively, the P450 enzymes can be purified and reconstituted with coenzymes into artificial membranes. 

Another major factor when considering whole-cell versus cell-free reactions are the overall reaction kinetics. Some enzymatic reactions utilize a complex multicomponent enzyme system. Reconstitution of the crude or purified enzyme components are not usually as effective in vitro as they are when they remain in the intracellular milieu. Whole cells have often been called little bags of enzymes. Although this is an oversimplification, it is a useful concept to consider. Whole cells sequester the enzyme components in a small but concentrated form, which is usually optimal for high efficiency. Whole cells also contain co-factors, including the systems that recycle them, and control pH and ionic strength. Altogether these factors combine to make whole cells a very useful form for the presentation and use of sensitive enzyme catalysts. 

A reaction mixture containing IMP, aspartic acid, and GTP was prepared, and the reaction was started by the addition of purified sAMP synthetase. As the incubation proceeded, samples were removed and analyzed by HPLC. As shown in Figure 10.9, the chromatograms reveal the presence of the substrates and the formation of sAMP. After incubation for 20 minutes, the multienzyme system was reconstituted by the addition of a sample of sAMP lyase to the reaction. The reconstituted system was incubated for an additional 10 minutes, and a sample removed at that time was analyzed by HPLC. The profile (Fig. 10.9) illustrates a decline in the level of sAMP and the appearance of a new peak, AMP, confirming the successful reconstitution of this two-enzyme system. 

Lotlikar PD, Baldy WJ Jr, Dwyer EN. 1975. Dimethylnitrosamine demethylation by reconstituted liver microsomal cytochrome P-450 enzyme system. Biochem J 152 705-708. 

Purification and reconstitution of the enzyme system will be required before elucidation of the mechanism of its activation. 

The evidence to support the idea that many, if not all, membrane proteins possess a immobile, long-lived annulus was of two kinds (a) biochemical and (b) spin-label studies. The first was based upon the fact that to maintain full enzyme activity, at least 30 lipid molecules per protein are required. The second was the presence of an immobile ESR signal at high protein to phospholipid content. Tanford has shown however that phospholipid is not essential for full enzymatic activity and that the hydrophobic fluid environment of a detergent is quite sufficient [75]. Furthermore, deuterium magnetic resonance studies on reconstituted ATPase systems have cast... 

Groves and co-workers have reported that the epoxidation of franj-l-deuterio-propylene by a reconstituted system with purified P-450lm2 proceeded with significant loss of the deuterium labels (778). Further, the epoxidation of propylene by this enzyme system in D2O afforded predominantly rran5-l-deuteriopropy-lene oxide. When NADPH/O2 was replaced with PhIO in the enzyme system, no incorporation of deuterium from D2O in recovered propylene was observed. 

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