Dehydrogenase EC

The oxidation of choline to betaine is catalyzed by two enzymes. First, choline is oxidized to betaine aldehyde by an enzyme which is found in mitochondria in membrane-bound form. This enzyme is believed to be a flavoprotein containing nonheme iron. Betaine aldehyde is then oxidized to betaine by a soluble enzyme, which is NAD-linked. Betaine aldehyde dehydrogenase appears to be present both in mitochondria and the soluble fraction of liver 243, 246). 

The existence of choline dehydrogenase was first demonstrated by Mann and Quastel in 1937 247, 248) in extracts of rat liver and kidney. These authors also obtained evidence that the first oxidation product of choline was betaine aldehyde. Others showed subsequently that choline oxidase activity resided in the mitochondrial fraction of rat liver and is linked to the respiratory chain 249, 250). Detergents 251, 252), solvent treatment of fragmented mitochondria 253), and venom phospholipase 254-256) have been used for extraction and solubilization of choline dehydrogenase. Among these, the best method reported to date appears to be the digestion of acetone-powdered mitochondria with venom phospholipase. Choline dehydrogenase, partially purified from phospholipase extracts of rat liver mitochondria, contains 1 mole of flavin and 4 g-atoms of nonheme iron per 850,000 g protein. The flavin is claimed to be acid- 

Information regarding the involvement of flavin and iron in enzyme catalysis is not available. Rothschild et al. (258) have reported that dialysis of rat liver particles resulted in the loss of choline-cytochrome c reductase activity, which could be restored by addition of FAD but not FMN. However, these results have not been substantiated by others (255). Singer has stated that the difference spectrum of the enzyme shows bleaching by substrate in both the flavin and the iron regions (227). This spectrum has not been published. 

It was shown by Strength et al. (261) that the oxidation of choline by a particulate preparation from rat liver was considerably enhanced upon addition of NAD. Others showed that choline oxidation by isolated rat liver mitochondria was completely inhibited by Amytal when oxygen, cytochrome c, ferricyanide, or methylene blue was the electron acceptor -264) Choline dehydrogenase activity of particles and soluble prepa- 

It has been shown that the oxidation of choline by isolated rat liver mitochondria is biphasic (269). The initial phase of choline oxidation is slow and coupled to the uptake of inorganic phosphate. The ensuing phase is 3-5 times faster and not coupled to phosphorylation. The slow phase can be extended in the presence of Mg-+ and ADP or ATP. These compounds are considered to control the permeability of mitochondria to choline (270). Calcium ions and conditions which result in mitochondrial swelling and membrane disruption have been shown to increase choline oxidation (266, 271). 

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Although alcohol dehydrogenases (ADH) also catalyze the oxidation of aldehydes to the corresponding acids, the rate of this reaction is significantly lower. The systems that combine ADH and aldehyde dehydrogenases (EC 1.2.1.5) (AldDH) are much more efficient. For example, HLAD catalyzes the enantioselective oxidation of a number of racemic 1,2-diols to L-a-hydroxy aldehydes which are further converted to L-a-hydroxy acids by AldDH (166). 

L-Sorbose + NADP+ = 5-dehydro-D-fmctose + NADPH (reaction of sorbose dehydrogenase, EC 1.1.1.123)... 

Figure 5. Example of dehydrogenase reactions which can be coupled with the bienzymatic bacterial bioluminescent system. ADH = alcohol dehydrogenase (EC 1.1.1.1), SDH = sorbitol dehydrogenase (EC 1.1.1.14), LDH = lactate dehydrogenase (EC 1.1.1.27), MDH = malate dehydrogenase (EC 1.1.1.37).

Figure 8.2 The effect of pH on the enzyme lactate dehydrogenase (EC 1.1.1.27). The enzyme shows maximum activity at pH 7.4 (A). When stored in buffer solutions with differing pH values for 1 h before re-assaying at pH 7.4, it shows complete recovery of activity from pH values between 5 and 9 but permanent inactivation outside these limits (B).

Figure 8.10 The quaternary structure of proteins. The enzyme lactate dehydrogenase (EC 1.1.1.27) has a relative molecular mass of approximately 140 000 and occurs as a tetramer produced by the association of two different globular proteins (A and B), a characteristic that results in five different hybrid forms of the active enzyme. The A and B peptides are enzymically inactive and are often indicated by M (muscle) and H (heart). The A4 tetramer predominates in skeletal muscle while the B4 form predominates in heart muscle but all tissues show most types in varying amounts.

The reaction mixture for a coupled assay includes the substrates for the initial or test enzyme and also the additional enzymes and reagents necessary to convert the product of the first reaction into a detectable product of the final reaction. The enzyme aspartate aminotransferase (EC 2.6.1.1), for instance, results in the formation of oxaloacetate, which can be converted to malic acid by the enzyme malate dehydrogenase (EC 1.1.1.37) with the simultaneous conversion of NADH to NAD+, a reaction which can be followed spectropho-tometrically at 340 nm ... 

Many assays have been described in which the initial product forms the substrate of an intermediary reaction involving auxiliary enzymes. The assay of creatine kinase (EC 2.13.2), for example, involves hexokinase (EC 2.7.1.1) as the auxiliary enzyme and glucose-6-phosphate dehydrogenase (EC 1.1.1.49) as the indicator enzyme ... 

Reactions do not necessarily go to completion and regardless of the amount of enzyme used, the equilibrium position of the reaction will not change. It is important for quantitative measurements that the reaction goes as near to completion as possible and this may be achieved by a variety of methods. The equilibrium position may be altered by changing the pH away from the optimum for the enzyme. For example, the equilibrium position for the reaction in which pyruvate is converted to lactate by lactate dehydrogenase (EC 1.1.1.27) lies very much towards pyruvate at the normal pH of 7.6 but at pH 9.0 the equilibrium is altered towards lactate. 

Very low concentrations of substrates may be assayed by recycling the test substrate for an appreciable but definite period of time and measuring the amount of product formed. The coenzyme NADPH, for instance, may be assayed using the two enzymes glutamate dehydrogenase (EC 1.4.1.3) and glucose-6-phosphate dehydrogenase (EC 1.1.1.49) ... 

Direct kinetic assays are the only valid methods for the measurement of activators and inhibitors and calibration plots of the percentage activation or inhibition by known amounts of the substance can be made. Examples of inhibition assays include the quantitation of organophosphorus pesticides using the inhibition of cholinesterase (EC 3.1.1.7) while manganese can be measured in amounts as low as 1 X 10-12 mol using its activating effect on isocitrate dehydrogenase (EC 1.1.1.41). 

L-Amino acid oxidase has been used to measure L-phenylalanine and involves the addition of a sodium arsenate-borate buffer, which promotes the conversion of the oxidation product, phenylpyruvic acid, to its enol form, which then forms a borate complex having an absorption maximum at 308 nm. Tyrosine and tryptophan react similarly but their enol-borate complexes have different absorption maxima at 330 and 350 nm respectively. Thus by taking absorbance readings at these wavelengths the specificity of the assay is improved. The assay for L-alanine may also be made almost completely specific by converting the L-pyruvate formed in the oxidation reaction to L-lactate by the addition of lactate dehydrogenase (EC 1.1.1.27) and monitoring the oxidation of NADH at 340 nm. 

Some enzymes with improved single amino acid specificity are commercially available. An example is phenylalanine dehydrogenase (EC 1.4.1.1), derived from bacterial sources, which acts on phenylalanine with the simultaneous conversion of NAD to NADH. Quantitation of the phenylalanine is based on determining the amount of NADH produced using standard procedures. In the direct methods, the absorbance at 340 nm is measured, whereas in the colorimetric methods, the reaction is coupled to an electron acceptor... 

An alternative method uses glycerol dehydrogenase (EC 1.1.1.6) to produce NADH. This is measured either by its absorbance at 340 nm or by using a diaphorase (EC 1.6.4.3), which catalyses the reduction of a dye, p-iodonitro-tetrazolium violet (INT) to produce a coloured complex with an absorption maximum at 540 nm ... 

Aldehyde dehydrogenase (EC 1.2.1.3) catalyzes the oxidation of aldehydes to acids (see Sect. 3.7.2). The enzyme is ubiquitously distributed, but has mainly been characterized in brain and liver, where it is found in the cytoplasm, mitochondria, and microsomes. It is not clear whether its esterase activity has a physiological role or is a surviving activity inherited from an evolutionary thiolesterase precursor. 

Dihydro-1,2-dihydroxybenzene (10.13) is oxidized by dihydrodiol dehydrogenase (EC 1.3.1.20) to catechol (10.15) (Chapt. 4 in [la]) [76], In a typical experiment in which 10.13 is incubated with phenobarbital-induced rabbit liver microsomes, phenol (10.14), catechol (10.15), and hydroquinone (10.16) represent 54, 39, and 1%, respectively, of the total metabolites detected [75]. In other words, neither benzene oxide (10.1) nor its hydration product l,2-dihydro-l,2-dihydroxybenzene (10.13) was detected. 

This approach was coupled to a system of three NAD+-dependent enzymes comprised of alcohol dehydrogenase (EC 1.1.1.1), aldehyde dehydrogenase (EC 1.2.1.3), and formate dehydrogenase (EC 1.2.1.2) to create an electrode theoretically capable of complete oxidation of methanol to carbon dioxide, as shown in Eigure 5. The anode was, in turn, coupled to a platinum-catalyzed oxygen cathode to produce a complete fuel cell operating at pH 7.5. With no externally applied convection, the cell produced power densities of 0.67 mW/cm at 0.49 V for periods of less than 1 min, before the onset of concentration polarization. 

Aspartate kinase [EC 2.T.2.4], also known as asparto-kinase, catalyzes the reaction of aspartate with ATP to produce 4-phosphoaspartate and ADP. The enzyme isolated from E. coli is a multifunctional protein, also exhibiting the ability to catalyze the reaction of homoserine with NAD(P) to produce aspartate 4-semialdehyde and NAD(P)H (that is, the activity of homoserine dehydrogenase, EC 1.1.1.3). 

This enzyme complex [EC 1.2.4.4], also known as 3-methyl-2-oxobutanoate dehydrogenase (lipoamide) and 2-oxoisovalerate dehydrogenase, catalyzes the reaction of 3-methyl-2-oxobutanoate with lipoamide to produce S-(2-methylpropanoyl)dihydrolipoamide and carbon dioxide. Thiamin pyrophosphate is a required cofactor. The complex also can utilize (5)-3-methyl-2-oxopenta-noate and 4-methyl-2-oxopentanoate as substrates. The complex contains branched-cham a-keto acid decarboxylase, dihydrolipoyl acyltransferase, and dihydrolipoa-mide dehydrogenase [EC 1.8.1.4]. 

Yang and Schulz also formulated a treatment of coupled enzyme reaction kinetics that does not assume an irreversible first reaction. The validity of their theory is confirmed by a model system consisting of enoyl-CoA hydratase (EC 4.2.1.17) and 3-hydroxyacyl-CoA dehydrogenase (EC 1.1.1.35) with 2,4-decadienoyl coenzyme A as a substrate. Unlike the conventional theory, their approach was found to be indispensible for coupled enzyme systems characterized by a first reaction with a small equilibrium constant and/or wherein the coupling enzyme concentration is higher than that of the intermediate. Equations based on their theory can allow one to calculate steady-state velocities of coupled enzyme reactions and to predict the time course of coupled enzyme reactions during the pre-steady state. 

Glutamate dehydrogenase [EC 1.4.1.2] catalyzes the reaction of L-glutamate with NAD+ and water to produce a-ketoglutarate (or, 2-oxoglutarate), ammonia, and NADH. 

Glycerol-3-phosphate dehydrogenase [EC 1.1.99.5] is a flavoprotein that catalyzes the reaction of sn-glycerol 3-phosphate with an acceptor substrate to produce glycerone phosphate (dihydroxyacetone phosphate) and the reduced acceptor. 

NADH dehydrogenase (ubiquinone) [EC 1.6.5.3] (also called ubiquinone reductase, type I dehydrogenase, and complex I dehydrogenase) catalyzes the reaction of NADH with ubiquinone to produce NAD and ubiqui-nol. The complex, which uses EAD and iron-sulfur proteins as cofactors, is found in mitochondrial membranes and can be degraded to form NADH dehydrogenase [EC... 

I. 6.99.3]. NADH dehydrogenase [EC 1.6.99.3] catalyzes the reaction of NADH with an acceptor to produce NAD+ and the reduced acceptor. Iron-sulfur and flavo-proteins are still being used as cofactors with this component of EC 1.6.5.3. Interestingly, after certain preparations have been followed, cytochrome c may serve as the acceptor substrate. 

A major class of enzymes that catalyze oxidation-reduction reactions. This class includes dehydrogenases, reductases, oxygenases, peroxidases, and a few synthases. Examples include alcohol dehydrogenase (EC 1.1.1.1), aldehyde oxidase (EC 1.2.3.1), orotate reductase (EC 1.3.1.14), glutamate synthase (EC 1.4.1.14), NAD(P) transhydrogenase (EC 1.6.1.1), and glutathione peroxidase (EC 1.11.1.9). 

Prephenate dehydrogenase [EC 1.3.1.12] catalyzes the reaction of prephenate with NAD+ to produce 4-hydro-xyphenylpyruvate, carbon dioxide, and NADH. This enzyme in enteric bacteria also possesses chorismate mutase activity and converts chorismate into prephenate. Prephenate dehydrogenase (NADP+) [EC 1.3.1.13] catalyzes the reaction of prephenate with NADP+ to produce 4-hydroxyphenylpyruvate, carbon dioxide, and NADPH. 

Pyruvate dehydrogenase (lipoamide) [EC 1.2.4.1], which requires thiamin pyrophosphate, catalyzes the reaction of pyruvate with lipoamide to produce 5-acetyldihydroli-poamide and carbon dioxide. It is a component of the pyruvate dehydrogenase complex (which also includes dihydrolipoamide dehydrogenase [EC 1.8.1.4] and dihy-drolipoamide acetyltransferase [EC 2.3.1.12]). Pyruvate dehydrogenase (cytochrome) [EC 1.2.2.2] catalyzes the... 

Succinate dehydrogenase (ubiquinone) [EC 1.3.5.1], a multiprotein complex found in the mitochondria, catalyzes the reaction of succinate with ubiquinone to produce fumarate and ubiqumol. The enzyme requires FAD and iron-sulfur groups. It can be degraded to form succinate dehydrogenase [EC 1.3.99.1], a FAD-dependent system that catalyzes the reaction of succinate with an acceptor to produce fumarate and the reduced acceptor, but no longer reacts with ubiquinone. 

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