Dimeric dihydrodiol dehydrogenase in monkey kidney. Substrate specificity, stereospecificity of hydrogen transfer, and distribution

Chemical cross-linking and NADPH binding studies suggested that the native dihydrodiol dehydrogenase from monkey kidney is a basic dimer having a molecular weight of 78,000 and one active site per the subunit. The enzyme oxidized specifically trans-dihydrodiols of benzene and naphthalene, whereas it catalyzed the reduction of dihydroxyacetone and dihydroxyacetone phosphate at a physiological pH, 7.4. The Km and kcat values for dihydroxyacetone phosphate were 5.0 mM and 4.3 s-1, respectively. The enzyme transferred the 4-pro-R hydrogen atom of NADPH to the carbonyl substrate. Immunochemical experiments using an antibody against the dimeric enzyme revealed the specific distribution of the enzyme in the kidney of this animal. By immunohistochemical staining with the specific antibody, the immunoreactivity was found in proximal and distal tubules of the cortex, and in the loop of Henle of the medulla.     

Reactions at prochiral centers. Interdependence in the estimation of enzyme stereospecificity toward prochiral centers and the configurational purity of labeled substrates.

In many enzymic reactions one or the other hydrogen is released from a prochiral center abCH2. To determine the stereospecificity with respect to such centers under nonreversal conditions a substrate with a chiral center abC1HH is employed (H = 3H or 2H). Assuming the simplest possible model, the R specificity of an enzyme or enzyme system (eR) may be defined in terms of the configurational R purity of the substrate with respect to the labeled center (pR), and the fraction of H released (r) or conserved (l - r) during the reaction by the equation eR = [(l - r) -pR]/[1 - 2pR]. Where r is constant, this is a rectangular hyperbola. The apparent stereospecificity calculated with this equation serves as a characteristic of the experimental system irrespective of the assumed model. The problems arising from direct isotope effects are discussed and the theoretical advantages of using product 3H:14C ratios under low conversion conditions noted. Applications of the equation to the determination of configurational purity at labeled centers, to the study of stereospecificity in enzymes and enzyme systems, and to the study of the biosynthesis of natural products are discussed.     

Stereospecificity of hydrogen transfer in the saccharopine dehydrogenase reaction.

The stereospecificity of hydrogen transfer in the synthesis of saccharopine from alpha-ketoglutarate and L-lysine catalyzed by saccharopine dehydrogenase (N5-(1,3-dicarboxypropyl)-L-lysine: NAD oxidoreductase (L-lysine-forming), EC 1.5.1.7) was examined by using [4A-3H]- and [4B-3H]NADH. The enzyme showed the A-stereospecificity. The NMR analysis of the saccharopine prepared with [4"A-2H]NADH revealed that the label was incorporated into the C-2 of the glutaryl moiety.     

Sensitive non-radioactive determination of aminotransferase stereospecificity for C-4' hydrogen transfer on the coenzyme

A sensitive non-radioactive method for determination of the stereospecificity of the C-4′ hydrogen transfer on the coenzymes (pyridoxal phosphate, PLP; and pyridoxamine phosphate, PMP) of aminotransferases has been developed. Aminotransferase of unknown stereospecificity in its PLP form was incubated in ²H₂O with a substrate amino acid resulted in PMP labeled with deuterium at C-4′ in the pro-S or pro-R configuration according to the stereospecificity of the aminotransferase tested. The [4′-²H]PMP was isolated from the enzyme protein and divided into two portions. The first portion was incubated in aqueous buffer with apo-aspartate aminotransferase (a reference si-face specific enzyme), and the other was incubated with apo-branched-chain amino acid aminotransferase (a reference re-face specific enzyme) in the presence of a substrate 2-oxo acid. The ²H at C-4′ is retained with the PLP if the aminotransferase in question transfers C-4′ hydrogen on the opposite face of the coenzyme compared with the reference aminotransferase, but the ²H is removed if the test and reference aminotransferases catalyze hydrogen transfer on the same face. PLP formed in the final reactions was analyzed by LC–MS/MS for the presence or absence of ²H. The method was highly sensitive that for the aminotransferase with ca. 50 kDa subunit molecular weight, only 2 mg of the enzyme was sufficient for the whole test. With this method, the use of radioactive substances could be avoided without compromising the sensitivity of the assay.     

Stereospecificity of hydrogen transfer by phosphoglycerate dehydrogenase.

Phosphoglycerate dehydrogenase (EC 1.1.1.95) has been shown to be A site specific in its hydrogen transfer capacity unlike other dehydrogenases which use phosphorylated substrates. The experiments have been carried out using a coupled assay system with yeast alcohol dehydrogenase. The specific activity measurements of the reaction products indicate the possible influence of an isotope effect on this system.     

Direct transfer of NADH from malate dehydrogenase to Complex I in Escherichia coli

During aerobic growth of Escherichia coli, nicotinamide adenine dinucleotide (NADH) can initiate electron transport at either of two sites: Complex I (NDH-1 or NADH: ubiquinone oxidoreductase) or a single-subunit NADH dehydrogenase (NDH-2). We report evidence for the specific coupling of malate dehydrogenase to Complex I. Membrane vesicles prepared from wild type cultures retain malate dehydrogenase and are capable of proton translocation driven by the addition of malate+NAD. This activity was inhibited by capsaicin, an inhibitor specific to Complex I, and it proceeded with deamino-NAD, a substrate utilized by Complex I, but not by NDH-2. The concentration of free NADH produced by membrane vesicles supplemented with malate+NAD was estimated to be 1 μM, while the rate of proton translocation due to Complex I was consistent with a some what higher concentration, suggesting a direct transfer mechanism. This interpretation was supported by competition assays in which inactive mutant forms of malate dehydrogenase were able to inhibit Complex I activity. These two lines of evidence indicate that the direct transfer of NADH from malate dehydrogenase to Complex I can occur in the E. coli system.     

The stereospecificity of the enzymic reduction of 21-dehydrocortisol by NADH.

(21R)-[21-3H]cortisol and (21S)-[21-3H]cortisol were synthesized by reduction of 21-dehydrocortisol by NADH in the presence of 21-hydroxysteroid dehydrogenase. The stereochemistry at carbon 21 was established after cleaving the side chain and oxidizing the resulting two epimers of tritiated glycolate with glycolate oxidase of known (2-pro-S) stereospecificity. From the distribution of radioactivity in the water and glyoxylate produced in this reaction, it was concluded that the reaction of 21-dehydrocortisol with (4S)-[4-3H]NADH catalyzed by 21-hydroxysteroid dehydrogenase results in a transfer of tritium from the 4S position of the nucleotide to form (21S)-[21-3H]cortisol, and that (21R)-[21-3H]cortisol resulted from the enzyme-catalyzed reduction of 21-dehydro[21-3H]cortisol with NADH. Nuclear magnetic resonance studies on both epimers at position 21 of [21-2H]cortisol and of [21-2H]cortisone prepared enzymically identify the transferring 21-pro-S hydrogen as the relatively downfield of the two 21-hydrogen atoms.     

A simple method for the rapid determination of the stereospecificity of NAD-dependent dehydrogenases applied to mammalian IMP dehydrogenase and bacterial NADH peroxidase

The stereospecificity of IMP dehydrogenase (IMP:NAD+ oxidoreductase, EC 1.1.1.205) from two different sources was determined. The enzyme preparations were obtained from murine lymphoblasts and from Escherichia coli. Both enzymes transferred the 2-3H of IMP to the pro-S position of carbon atom C-4 of the nicotinamide ring in NAD. Thus, B-sided stereospecificity is common to the enzyme from two very different species. In addition, the studies described here demonstrate that alcohol dehydrogenase and NADH peroxidase, used as auxiliary enzymes, in combination with a microdistillation procedure, should permit rapid determination of the stereospecificity of any NAD-dependent dehydrogenase for which the appropriate tritiated substrate is available.     

NMR studies of the stereospecificity of 20 -hydroxysteroid dehydrogenase

We (3,4) previously observed the reduction of 21-dehydrocorticosteroids in the presence of 20β-hydroxysteroid dehydrogenase proceeded at a faster rate than the reduction of the corresponding corticosteroids. The presence of adjacent carbonyl groups suggested the possibility that the increased rate of reduction of the 20-one,21-a1 steroid analogs resulted from a lack of specificity of the enzyme 20β-hydroxysteroid dehydrogenase for either the aldehyde or ketone group. Nuclear magnetic resonance spectroscopy indicated that the angular methyl groups of the steroid were sensitive probes for the constituents on the basic steroid skeleton. The C₁₈ methyl resonance of 17,21-dihydroxy-4-pregnene-3,20-dione and 17-hydroxy-3,20-dioxo-4-pregnene-21-a1 were 0.722 ppm and 0.728 ppm respectively. The magnitude and sign of the change in chemical shift of the C₁₈ methyl resonance for the enzymatic products of 17,21-dihydroxy-4-pregnene-3,20-dione and 17-hydroxy-3,20-dioxo-4-pregnene-21-a1 (+0.135 ppm and +0.144 ppm respectively) were consistent with a stereochemical assignment of 20β-hydroxyl.     

Lactate dehydrogenase in Phycomyces blakesleeanus.

1. An NAD-specific L(+)-lactate dehydrogenase (EC 1.1.1.27) from the mycelium of Phycomyces blakesleeanus N.R.R.L. 1555 (-) was purified approximately 700-fold. The enzyme has a molecular weight of 135,000-140,000. The purified enzyme gave a single, catalytically active, protein band after polyacrylamide-gel electrophoresis. It shows optimum activity between pH 6.7 and 7.5. 2. The Phycomyces blakesleeanus lactate dehydrogenase exhibits homotropic interactions with its substrate, pyruvate, and its coenzyme, NADH, at pH 7.5, indicating the existence of multiple binding sites in the enzyme for these ligands. 3. At pH 6.0, the enzyme shows high substrate inhibition by pyruvate. 3-hydroxypyruvate and 2-oxovalerate exhibit an analogous effect, whereas glyoxylate does not, when tested as substrates at the same pH. 4. At pH 7.5, ATP, which inhibits the enzyme, acts competitively with NADH and pyruvate, whereas at pH 6.0 and low concentrations of ATP it behaves in a allosteric manner as inhibitor with respect to NADH, GTP, however, has no effect under the same experimental conditions. 5. Partially purified enzyme from sporangiophores behaves in entirely similar kinetic manner as the one exhibited by the enzyme from mycelium.