Reconstituted enzyme

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 al., unpublished results).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-... 

Flavin Coenzymes.—5-Deazaflavin-adenine dinucleotide (2) can be prepared from the 5-deazaFMN,21 using a FAD pyrophosphorylase from rat liver.22 When the apoprotein of D-amino-acid oxidase from pig kidney is reconstituted with (2), no oxidation of D-alanine is observed, although the flavin chromophore in the reconstituted enzyme is reduced on the addition of DL-amino-acids.22 This has been interpreted as indicating that hydrogen transfer from the amino-acid to (2) can still... 

A number of cases are known in which the properties of an enzyme are markedly altered by interaction with a membrane. Of course, in some cases the normal function of an enzyme is destroyed when it is removed from the membrane. For example, the mitochondrial coupling factor cannot synthesize ATP when removed from the membrane, since coupling to a proton gradient is required. The portion of the coupling factor that is easily solubilized (F,) is an ATPase. The steady-state kinetic properties of this solubilized ATPase are appreciably changed when it is reconstituted with mitochondrial membranes The turnover numbers and pH dependencies are different the solubilized enzyme is strongly inhibited by ADP, whereas the reconstituted enzyme is not and the reconstituted enzyme is inhibited by oligomycin, whereas the solubilized enzyme is not. 

In general, the strength of substrate binding to membrane-bound enzymes spans a range similar to that encountered with soluble enzymes. The presence of a membrane can either enhance or inhibit substrate binding. The solubilized ATPase and the reconstituted enzyme have only slightly different Michaelis constants. 

The Fc-HRP activity was quantified using two different substrates of HRP, i.e., ABTS and water-soluble ferrocene derivatives. Rate laws and kinetic parameters for native HRP and Fc-HRP have been compared. The native and the reconstituted enzymes catalyze the oxidation of ABTS in accordance with the Michaelis-Menten kinetics the inverse rate versus [ABTS]-1 plots are linear and the values of the maximum rates Vm and the Michaelis constant Km are summarized... 

MacIntyre and Dean (119) report that acid phosphatase from D. melanogaster has slow and fast electrophoretic variants specified by co-dominant alleles. Thus, acid phosphatases AA, BB, and AB were studied. Types AA and BB could be inactivated by exposure to acid. Reactivation of enzymic activity could be accomplished by dialysis against buffers at pH 6.5. Mixtures of AA and BB produced some AB reconstituted enzyme. From this evidence it seems very probable that acid phosphatase, at least in this species, consists of at least two polypeptide chains. 

The Anfinsen experiment raises the question Must we always use x-ray diffraction to determine a protein s structure In the case of ribonuclease the structure is known from x-ray diffraction, but at the time of Anfinsen s investigation it was not. He relied heavily on the fact that the reconstituted enzyme had the same activity as the native enzyme. This remains an acceptable approach as long as one is working with an enzyme that has a demonstrable catalytic activity, but what of the many proteins that do not ... 

A further approach to controlling electrical communication between redox proteins and their electrode support through a photo-command interface includes photo stimulated electrostatic control over the electrical contact between the redox enzyme and the electrode in the presence of a diffusional electron mediator (Scheme 12).[58] A mixed monolayer, consisting of the photoisomerizable thiolated nitrospiropyran units 30 and the semi-synthetic FAD cofactor 25, was assembled on an Au electrode. Apo-glucose oxidase was reconstituted onto the surface FAD sites to yield an aligned enzyme-layered electrode. The surface-reconstituted enzyme (2 x 10-12 mole cm-2) by itself lacked electrical communication with the electrode. In the presence of the positively charged, protonated diffusional electron mediator l-[l-(dimethylamino)ethyl]ferrocene 29, however, the bioelectrocatalytic functions of the enzyme-layered electrode could be activated and controlled by the photoisomerizable component co-immobilized in the monolayer assembly (Figure 12). In the... 

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. 

Preliminary studies in our laboratory with orcinol hydroxylase, from a pseudomonad, suggested that the 1-dFAD-reconstituted enzyme would catalyze NADH oxidation, but 02 was reduced exclusively to H202. No oxygen transfer yielding trihydroxytoluene could be detected. Because of instability of both trihydroxytoluene oxygenation product and orcinol hydroxylase apoenzyme, the 1-dFAD studies were pursued next in collaboration with Massey, Husain, Ballou, and Entsch at University of Michigan with the most well characterized bacterial flavoenzyme hydroxylase, p-hydroxybenzoate hydroxylase (2, 26, 27). The apoenzyme was reconstituted stably with 1-deazaFAD... 

Furthermore, the reconstituted enzyme has the same allosteric properties as the native enzyme. Thus, ATCase is composed of discrete catalytic and regulatory suhunits, which interact in the native enzyme to produce its allosteric behavior. 

It also was found that the direction of the photobiocatalytic switch of the nitrospiropyran-FAD-reconstituted enzyme is controlled by the electrical properties of the electron transfer mediator. With ferrocene dicarboxylic acid as a diffusional electron transfer mediator, the enzyme in the nitrospiropyran-FAD state (10a) was found to correspond to the OFF state bio-catalyst, while the protonated nitromerocyanine state of the enzyme (10b) exhibits ON behavior. In the presence of the protonated 1-[1-(dimethyl-amino )ethyl]ferrocene, the direction of the photobioelectrocatalytic switch is reversed. The nitrospiropyran-enzyme state (10a) is activated toward the electrocatalyzed ox idation of glucose, while the protonated nitromerocyanine enzyme state (10b) is switched OFF, and is inactive for the electrochemical oxidation of glucose. This control of the photoswitch direction of the photoisomerizable reconstituted enzyme was attributed to electrostatic interactions between the diffusional electron mediator and the photoisomefizable unit... 

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