Effects of metal ions on the substrate-specificity and activity of proton-pumping nicotinamide nucleotide transhydrogenase from Escherichia coli

Nicotinamide nucleotide transhydrogenase catalyzes the reversible reduction of NADP+ by NADH and a concomitant proton translocation. It was demonstrated (Glavas, N.A. and Bragg, P.D. (1995) Biochim. Biophys. Acta 1231, 297-303) that the Escherichia coli transhydrogenase also catalyzed a reduction of the NAD-analogue 3-acetylpyridine-NAD+ (AcPyAD+) by NADH at low pH and in the absence of (added) NADP(H) and high salt concentrations The mechanism of this reaction has as yet not been explained. In the present study, the E. coli transhydrogenase was purified by affinity chromatography through the NADP(H)-site, rendering the pure enzyme free of NADP(H). Using this preparation it was confirmed that the enzyme readily catalyzes the above reaction. Inhibitors specific for the NADP(H)-site, e.g., palmitoyl-Coenzyme A and adenosine-2'-monophosphate-5'-diphosphoribose, strongly inhibited the reduction of AcPyAD+ by NADH, whereas an inhibitor of the NAD(H)-site, adenosine 5'-diphosphoribose, was less inhibitory. This suggests that a lack of metal ions or other ions at low pH induces an unspecific interaction of the NADP(H)-site with AcPyAD+ or NADH, presumably NADH, producing a cyclic reduction of AcPyAD+ by NADH via NAD(H) bound in the NADP(H) site. A stimulation of reduction of AcPyAD+ by NADPH by Mg2+ present during reconstitution of transhydrogenase in phospholipid vesicles was observed, but it is presently unclear whether this effect is related to that seen with the detergent-dispersed enzyme.     

Altered cell shape is linked to increased p34cdc2 gene expression in fibroblasts expressing a mutant E2F-1 transcription factor

Transhydrogenase is a proton pump. It has separate binding sites for NAD+/NADH (on domain I of the protein) and for NADP+/NADPH (on domain III). Purified, detergent-dispersed transhydrogenase from Escherichia coli catalyses the reduction of the NAD+ analogue, acetylpyridine adenine dinucleotide (AcPdAD+), by NADH at a slow rate in the absence of added NADP+ or NADPH. Although it is slow, this reaction is surprising, since transhydrogenase is generally thought to catalyse hydride transfer between NAD(H)--or its analogues and NADP(H)--or its analogues, by a ternary complex mechanism. It is shown that hydride transfer occurs between the 4A position on the nicotinamide ring of NADH and the 4A position of AcPdAD+. On the basis of the known stereospecificity of the enzyme, this eliminates the possibilities of transhydrogenation(a) from NADH in domain I to AcPdAD+ wrongly located in domain III; and (b) from NADH wrongly located in domain III to AcPdAD+ in domain I. In the presence of low concentrations of added NADP+ or NADPH, detergent-dispersed E. coli transhydrogenase catalyses the very rapid reduction of AcPdAD+ by NADH. This reaction is cyclic; it takes place via the alternate oxidation of NADPH by AcPdAD+ and the reduction of NADP+ by NADH, while the NADPH and NADP+ remain tightly bound to the enzyme. In the present work, it is shown that the rate of the cyclic reaction and the rate of reduction of AcPdAD+ by NADH in the absence of added NADP+/NADPH, have similar dependences on pH and on MgSO4 concentration and that they have a similar kinetic character. It is therefore suggested that the reduction of AcPdAD+ by NADH is actually a cyclic reaction operating, either with tightly bound NADP+/NADPH on a small fraction (< 5%) of the enzyme, or with NAD+/NADH (or AcPdAD+/AcPdADH) unnaturally occluded within the domain III site. Transhydrogenase associated with membrane vesicles (chromatophores) of Rhodospirillum rubrum also catalyses the reduction of AcPdAD+ by NADH in the absence of added NADP+/NADPH. When the chromatophores were stripped of transhydrogenase domain I, that reaction was lost in parallel with 'normal reverse' transhydrogenation (e.g., the reduction of AcPdAD+ by NADPH). The two reactions were fully recovered upon reconstitution with recombinant domain I protein. However, after repeated washing of the domain I-depleted chromatophores, reverse transhydrogenation activity (when assayed in the presence of domain I) was retained, whereas the reduction of AcPdAD+ by NADH declined in activity. Addition of low concentrations of NADP+ or NADPH always supported the same high rate of the NADH-->AcPdAD+ reaction independently of how often the membranes were washed. It is concluded that, as with the purified E. coli enzyme, the reduction of AcPdAD+ by NADH in chromatophores is a cyclic reaction involving nucleotides that are tightly bound in the domain III site of transhydrogenase. However, in the case of R. rubrum membranes it can be shown with some certainty that the bound nucleotides are NADP+ or NADPH. The data are thus adequately explained without recourse to suggestions of multiple nucleotide-binding sites on transhydrogenase.     

Influence of vanadium on spin- and charge-density waves in chromium

Reduction of the antioxidant lipoic acid has been proposed to be catalyzed in vivo by lipoamide dehydrogenase (LipDH) or glutathione reductase (GR). We have found that thioredoxin reductase (TR) from calf thymus, calf liver, human placenta, and rat liver efficiently reduced both lipoic acid and lipoamide with Michaelis-Menten type kinetics in NADPH-dependent reactions. In contrast to LipDH, lipoic acid was reduced almost as efficiently as lipoamide. Under equivalent conditions at 20 degrees C, pH 8.0, mammalian TR reduced lipoic acid by NADPH 15 times more efficiently than the corresponding NADH dependent reduction catalyzed by LipDH (297 min-1 for TR vs. 20.3 min-1 for LipDH). Moreover, TR was 2.5 times faster in reducing lipoic acid with NADPH than in catalyzing the reverse reaction (oxidation of dihydrolipoic acid with NADP+). In contrast, LipDH was only 0.048 times as efficient in the forward reaction as compared to the reverse reaction (using NADH and NAD+). We conclude that all or part of the previously described NADPH-dependent lipoamide dehydrogenase (diaphorase) activities in mammalian systems should be attributed to TR. Our results suggest that in mammalian cells a significant part of the therapeutically important reduction of lipoic acid is catalyzed by thioredoxin reductase.     

Enantioselective sulfation of beta 2-receptor agonists by the human intestine and the recombinant M-form phenolsulfotransferase

The beta 2-receptor agonist class of drugs is metabolized in humans almost exclusively by sulfate conjugation. The objective of this investigation was to determine the influence of chemical structure on the stereoselectivity of the sulfoconjugation of these chiral drugs. The pure enantiomers of six beta 2-agonists, including those clinically most widely used, were all effectively sulfated both by the cytosol of the human intestine and the recombinant human M-form phenolsulfotransferase (PST). Whereas the apparent Km values (Km,app) for the sulfation of the individual drug enantiomers by the intestinal cytosol varied widely, ranging from 4.8 microM for (S)-isoproterenol to 889 microM for (S)-albuterol, these Km,app values were highly correlated with those obtained with M-PST (correlation coefficient 0.994). In contrast, the M-PST Vmax,app values were similar for all drug enantiomers, ranging from 276 to 914 pmol min-1 mg-1 protein, implying that substrate binding to M-PST by far is the main determinant of the sulfation activity. For isoproterenol, the Km,app for M-PST was 6.1 times higher for the active (R)- than for the inactive (S)-enantiomer. For other beta 2-agonists, the stereoselectivity decreased towards unity as the Km,app increased. However, for albuterol, containing a hydroxymethyl substituent at the aromatic ring, the stereoselectivity was dramatically reversed, with 10 times higher Km,app for the inactive (S)- than for the active (R)-enantiomer.     

Reactions catalyzed by tetrahydrobiopterin-free nitric oxide synthase

Murine macrophage nitric oxide synthase (NOS) was expressed in E. coli and purified in the presence (holoNOS) or absence (H4B-free NOS) of (6R)-tetrahydro-L-biopterin (H4B). Isolation of active enzyme required the coexpression of calmodulin. Recombinant holoNOS displayed similar spectral characteristics and activity as the enzyme isolated from murine macrophages. H4B-free NOS exhibited a Soret band at approximately 420 nm and, by analytical gel filtration, consisted of a mixture of monomers and dimers. H4B-free NOS catalyzed the oxidation of NG-hydroxy-L-arginine (NHA) with either hydrogen peroxide (H2O2) or NADPH and O2 as substrates. No product formation from arginine was observed under either condition. The amino acid products of NHA oxidation in both the H2O2 and NADPH/O2 reactions were determined to be citrulline and Ndelta-cyanoornithine (CN-orn). Nitrite and nitrate were also formed. Chemiluminescent analysis did not detect the formation of nitric oxide (*NO) in the NADPH/O2 reaction. The initial inorganic product of the NADPH/O2 reaction is proposed to be the nitroxyl anion (NO-) based on the formation of a ferrous nitrosyl complex using the heme domain of soluble guanylate cyclase as a trap, and the formation of a ferrous nitrosyl complex of H4B-free NOS during turnover of NHA and NADPH. NO- is unstable and, under the conditions of the reaction, is oxidized to nitrite and nitrate. At 25 degreesC, the H2O2-supported reaction had a specific activity of 120 +/- 14 nmol min-1 mg-1 and the NADPH-supported reaction had a specific activity of 31 +/- 6 nmol min-1 mg-1 with a KM,app for NHA of 129 +/- 9 microM. HoloNOS catalyzed the H2O2-supported reaction with a specific activity of 815 +/- 30 nmol min-1 mg-1 and the NADPH-dependent reaction to produce *NO and citrulline at 171 +/- 20 nmol min-1 mg-1 with a KM, app for NHA in the NADPH reaction of 36.9 +/- 0.3 microM.     

Esthetic problems in surgery of the male external genitalia: medico-legal aspects

A novel technique has been developed for semiquantitative detection of glutathione (GSH) in small volumes of liquid samples. GSH is detected via enzymatic linkage to the NADP/NADPH + H+ redox system through glutathione reductase. Accumulated NADPH is measured via the bioluminescent FMN oxidoreductase bacterial luciferase reaction. A linear correlation is obtained between bioluminescence intensity of the luciferase reaction and the GSH content of the liquid sample. Possible applications of this procedure are discussed.     

Polyol metabolism by a caries-conducive Streptococcus: purification and properties of a nicotinamide adenine dinucleotide-dependent mannitol-1-phosphate dehydrogenase

The mannitol-1-phosphate dehydrogenase (M1PDH) (EC 1.1.1.17) from Streptococcus mutans strain FA-1 was purified to approximately a 425-fold increase in specific activity with a 29% recovery of total enzyme units, using a combination of (i) streptomycin sulfate and ammonium sulfate precipitation and (ii) diethyl-aminoethyl-cellulose (DE-52), agarose A 0.5M, and agarose-nicotinamide adenine dinucleotide (NAD) affinity column chromatography. Polyacrylamide gel electrophoresis of the purified enzyme preparation showed a single protein component that coincided with a band of M1PDH activity. The enzyme had a molecular weight of approximately 45,000 and was stable for long periods of time when stored at -80 degrees C in the presence of beta-mercaptoethanol. Its activity was not affected by mono- or divalent cations, and high concentrations of ethylenedia-minetetraacetic acid were not inhibitory. The M1PDH catalyzed both the NAD-dependent oxidation of mannitol-1-phosphate and the reduced NAD (NADH)-dependent reduction of fructose-6-phosphate. The forward reaction was highly specific for mannitol-1-phosphate and NAD, whereas the reverse reaction was highly specific for NADH and fructose-6-phosphate. The K(m) values for mannitol-1-phosphate and NAD were 0.15 and 0.066 mM, respectively, and the K(m) values for fructose-6-phosphate and NADH were 1.66 and 0.016 mM, respectively. The forward and reverse reactions catalyzed by the M1PDH from S. mutans appeared to be under cellular control. Both adenosine 5'-triphosphate and fructose-6-phosphate were negative effectors of the forward reaction, whereas adenosine 5'-diphosphate served as a negative effector of the reverse reaction catalyzed by the enzyme.     

The E6 and E7 genes of human papilloma virus-type 16 protect primary astrocyte cultures from injury

Nicotinamide nucleotide transhydrogenase constitutes a proton pump which links the NAD(H) and NADP(H) pools in the cell by catalyzing a reversible reduction of NADP+ by NADH. The recent cloning and characterization of several proton-pumping transhydrogenases show that they share a number of features. They are composed of three domains, i.e., the hydrophilic domains I and III containing the NAD(H)- and NADP(H)-binding sites, respectively, and domain II containing the transmembrane and proton-conducting region. When expressed separately, the two hydrophilic domains interact directly and catalyze hydride transfer reactions similar to those catalyzed by the wild-type enzyme. An extensive mutagenesis program has established several amino acid residues as important for both catalysis and proton pumping. Conformational changes mediating the redox-driven proton pumping by the enzyme are being characterized. With the cloned, well-characterized and easily accessible transhydrogenases from E. coli and Rhodospirillum rubrum at hand, the overall aim of the transhydrogenase research, the understanding of the conformationally driven proton pumping mechanism, is within reach.     

Interactions of reduced and oxidized nicotinamide mononucleotide with wild-type and alphaD195E mutant proton-pumping nicotinamide nucleotide transhydrogenases from Escherichia coli

The interaction of reduced nicotinamide mononucleotide (NMNH), constituting one half of NADH, with the wild-type and alphaD195E proton-pumping nicotinamide nucleotide transhydrogenase from Escherichia coli was investigated. Reduction of thio-NADP+ by NMNH was catalysed at approximately 30% of the rate with NADH. Other activities including proton pumping and the cyclic reduction of 3'-acetyl-pyridine-NAD+ by NMNH in the presence of NADP+ were more strongly inhibited. The alphaD195 residue is assumed to interact with the 2'-OH moiety of the adenosine-5'-phosphate, i.e., the second nucleotide of NADH. Mutation of this residue to alphaD195E resulted in a 90% decrease in activity with NMNH as well as NADH as substrate, suggesting that it produced global structural changes of the NAD(H) binding site. The results suggest that the NMN moiety of NADH is a substrate of transhydrogenase, and that the adenine nucleotide is not required for catalysis or proton pumping.     

Dopaminergic activity and the discriminative stimulus effects of mu opioids in pigeons: importance of training dose and attenuation by the D3 agonist (+/-)-7-OH-DPAT

Chlorophyll fluorescence measurements were performed on osmotically lysed potato chloroplasts in order to characterize the reactions involved in the dark reduction of photosynthetic inter-system chain electron carriers. Addition of NADH or NADPH to lysed chloroplasts increased the chlorophyll fluorescence level measured in the presence of a non-actinic light until reaching Fmax, thus indicating an increase in the redox state of the plastoquinone (PQ) pool. The fluorescence increase was more pronounced when the experiment was carried out under anaerobic conditions and was about 50% higher when NADH rather than NADPH was used as an electron donor. The NAD(P)H-PQ oxidoreductase reaction was inhibited by diphenylene iodonium, N-ethylmaleimide and dicoumarol, but insensitive to rotenone, antimycin A and piericidin A. By comparing the substrate specificity and the inhibitor sensitivity of this reaction to the properties of spinach ferredoxin-NADP+-reductase (FNR), we infer that FNR is not involved in the NAD(P)H-PQ oxidoreductase activity and conclude to the participation of rotenone-insensitive NAD(P)H-PQ oxidoreductase. By measuring light-dependent oxygen uptake in the presence of DCMU, methyl viologen and NADH or NADPH as an electron donors, the electron flow rate through the NAD(P)H-PQ oxidoreductase is estimated to about 160 nmol O2 min-1 mg-1 chlorophyll. The nature of this enzyme is discussed in relation to the existence of a thylakoidal NADH dehydrogenase complex encoded by plastidial ndh genes. Copyright 1998 Elsevier Science B.V.     

Immunohistochemical localization of G protein beta1, beta2, beta3, beta4, beta5, and gamma3 subunits in the adult rat brain

The regional distributions of the G protein beta subunits (Gbeta1-beta5) and of the Ggamma3 subunit were examined by immunohistochemical methods in the adult rat brain. In general, the Gbeta and Ggamma3 subunits were widely distributed throughout the brain, with most regions containing several Gbeta subunits within their neuronal networks. The olfactory bulb, neocortex, hippocampus, striatum, thalamus, cerebellum, and brainstem exhibited light to intense Gbeta immunostaining. Negative immunostaining was observed in cortical layer I for Gbeta1 and layer IV for Gbeta4. The hippocampal dentate granular and CA1-CA3 pyramidal cells displayed little or no positive immunostaining for Gbeta2 or Gbeta4. No anti-Gbeta4 immunostaining was observed in the pars compacta of the substantia nigra or in the cerebellar granule cell layer and Purkinje cells. Immunoreactivity for Gbeta1 was absent from the cerebellar molecular layer, and Gbeta2 was not detected in the Purkinje cells. No positive Ggama3 immunoreactivity was observed in the lateral habenula, lateral septal nucleus, or Purkinje cells. Double-fluorescence immunostaining with anti-Ggamma3 antibody and individual anti-Gbeta1-beta5 antibodies displayed regional selectivity with Gbeta1 (cortical layers V-VI) and Gbeta2 (cortical layer I). In conclusion, despite the widespread overlapping distributions of Gbeta1-beta5 with Ggamma3, specific dimeric associations in situ were observed within discrete brain regions.     

Purification and biochemical properties of allelic forms of cytoplasmic glycerol-3-phosphate dehydrogenase from Drosophila virilis

Three homozygous allelic forms (alpha GPDHf, alpha GPDHm and alpha GPDHs) of NAD+-dependent glycerol-3-phosphate dehydrogenase (sn-glycerol-3-phosphate:NAD+ 2-oxidoreductase, EC 1.1.1.8) of Drosophila virilis were purified to homogeneity and their biochemical properties were compared. Although these three forms were mutually distinguishable by electrophoresis, no significant differences were found with respect to pH optima for both forward and reverse reactions (pH 6.0--6.5 for dihydroxyacetone phosphate reduction; pH 10.0--10.5 for glycerol 3-phosphate oxidation), native and subunit molecular weights (65 000 for native form; 35 000--37 000 for subunit) and Michaelis constants for NADH, glycerol 3-phosphate and NAD+ (5.3--6.0 microM for NADH; 1.8--1.9 mM for glycerol 3-phosphate; 100--110 microM for NAD+). Significant differences among three forms were observed in thermostability at 35 degrees C and inhibition by excess of dihydroxyacetone phosphate. The alpha GPDHf form was found to be most thermolabile and the alpha GPDHs form most susceptible to the inhibition.     

Purification and properties of NADH-dependent 5, 10-methylenetetrahydrofolate reductase (MetF) from Escherichia coli

A K-12 strain of Escherichia coli that overproduces methylenetetrahydrofolate reductase (MetF) has been constructed, and the enzyme has been purified to apparent homogeneity. A plasmid specifying MetF with six histidine residues added to the C terminus has been used to purify histidine-tagged MetF to homogeneity in a single step by affinity chromatography on nickel-agarose, yielding a preparation with specific activity comparable to that of the unmodified enzyme. The native protein comprises four identical 33-kDa subunits, each of which contains a molecule of noncovalently bound flavin adenine dinucleotide (FAD). No additional cofactors or metals have been detected. The purified enzyme catalyzes the reduction of methylenetetrahydrofolate to methyltetrahydrofolate, using NADH as the reductant. Kinetic parameters have been determined at 15 degreesC and pH 7.2 in a stopped-flow spectrophotometer; the Km for NADH is 13 microM, the Km for CH2-H4folate is 0.8 microM, and the turnover number under Vmax conditions estimated for the reaction is 1,800 mol of NADH oxidized min-1 (mol of enzyme-bound FAD)-1. NADPH also serves as a reductant, but exhibits a much higher Km. MetF also catalyzes the oxidation of methyltetrahydrofolate to methylenetetrahydrofolate in the presence of menadione, which serves as an electron acceptor. The properties of MetF from E. coli differ from those of the ferredoxin-dependent methylenetetrahydrofolate reductase isolated from the homoacetogen Clostridium formicoaceticum and more closely resemble those of the NADH-dependent enzyme from Peptostreptococcus productus and the NADPH-dependent enzymes from eukaryotes.     

Sample pretreatment with nitrate reductase and glucose-6-phosphate dehydrogenase quantitatively reduces nitrate while avoiding interference by NADP+ when the Griess reaction is used to assay for nitrite

An assay for the simultaneous measurement of nitrite and nitrate, products of nitric oxide metabolism, is described. Others have reported pretreating sample by using nitrate reductase (NR) and NADPH to reduce endogenous NO3- before assaying the resultant NO2- using the Griess reaction. However, we found that the NADP+ formed during pretreatment interfered with the Griess reaction when NADPH was used at concentrations necessary to drive the NR reaction. For instance, 500 microM NADP+ in 100 microM NaNO3- (without NR) causes a 90% interference with the formation of Griess reaction product. To limit interference, we modified the method by decreasing the NADPH concentration to 1 microM. NADPH was regenerated by coupling the NR reaction with that catalyzed by glucose-6-phosphate dehydrogenase (GD). Using this method, NaNO3- standard curves were linear up to 100 microM and coincided with control curves obtained using NaNO2- incubated in parallel. Addition of urine up to a strength of 20% did not interfere with the assay. Comparison with an alternative assay based on cadmium reduction resulted in the following linear regression: [Cd method] = 0.915*[NR-GD method] + 0.37, r2 = 0.997. Coupling GD to NR to recycle NADPH allows this cofactor to be used at a low concentration so that interference with the Griess reaction is negligible.     

The effects of reverse micelles on the reaction mechanism of alpha-chymotrypsin

Reverse micelles were employed to test the accuracy of the widely accepted mechanism for alpha-chymotrypsin in a highly structured aqueous system similar to intracellular conditions. Results yielded from spectrophotometrical assays of the alpha-chymotrypsin catalyzed hydrolysis of both p-nitrophenyl acetate (p-NPA) and p-nitrophenyl trimethylacetate (p-NPTA) were kinetically analyzed to determine constants typical of the proposed mechanistic model. This was accomplished through the establishment of a control, i.e. the well studied buffer system, for comparison between the reverse micellular environment and a bulk aqueous solution. Control group results yielded kinetic constants in favor of the proposed mechanism (Km = 1.55 x 10(-5) +/- 1.40 x 10(-6) M for p-NPA and a Km = 4.97 x 10(-6) +/- 2.29 x 10(-7) M, Km(app) = 4.92 x 10(-6) +/- 2.33 x 10(-8) M, k2 = 4.34 x 10(-3) +/- 1.31 x 10(-3), k(cat) = 1.96 x 10(-3) +/- 2.47 x 10(-4), and Ks = 1.60 x 10(-5) +/- 4.61 x 10(-6) M for p-NPTA). In contrast, similar reactions of the enzyme in a reverse micellular system produced kinetic constants atypical to that representative of the textbook mechanism. (Km = 1.59 x 10(-4) +/- 2.70 x 10(-5) M, Ks = -8.67 x 10(-5) +/- 4.46 x 10(-5) M and Km(app) = -4.80 x 10(-5) +/- 7.05 x 10(-5) M for p-NPA and Km = 1.95 x 10(-4) +/- 9.28 x 10(-5) M, Km(app) = -1.79 x 10(-4) +/- 2.36 x 10(-5) M, and Ks = -3.95 x 10(-4) +/- 1.18 x 10(-4) M for p-NPTA). In addition to negative kinetic constants, alpha-chymotrypsin seemed to display characteristics indicative of super-activity and a hysteretic response. Overall, the widely accepted mechanism for alpha-chymotrypsin appeared to fail within the confines of reverse micelles, due to the direct influence of the system's highly structured form.     

Flow cytometric assay of cytochemically demonstrated NAD(P)H oxidoreductase (diaphorase) activities

The tetrazolium salt 5-cyano-2,3-di-p-toluyl-tetrazolium chloride (CTC), yielding a fluorescent formazan on reduction, was used to measure NAD(P)H oxidoreductase activity. In this study, optimal conditions for the flow cytometric technique were determined empirically with tissue culture cell lines and mouse Ehrlich ascites cells. Applying a coupled reaction procedure, NADH and NADPH as substrates of the oxidoreductases to be measured are generated endogenously by lactate or glucose-6-phosphate dehydrogenase, respectively. The results were evaluated by combining spectrophotometry and flow cytometry. We obtained integral activities for each group of NADH and NADPH oxidoreductases. Furthermore, by counterstaining the DNA with DAPI, followed by bivariate analysis of flow cytometric data, our assay gives a detailed distribution of enzyme activities of all cells, even in subgroups present in heterogeneous cell populations. Therefore, this protocol permits the study of NAD(P)H oxidoreductase activities in ex vivo tumor samples in which mixed cellular populations may be present.     

Purification and characterization of virginiamycin M1 reductase from Streptomyces virginiae

Virginiamycin M1 (VM1), produced by Streptomyces virginiae, is a polyunsaturated macrocyclic lactone antibiotic belonging to the virginiamycin A group. S. virginiae possesses an activity which stereospecifically reduces a 16-carbonyl group of VM1, resulting in antibiotically inactive 16R-dihydroVM1. The corresponding VM1 reductase was purified to homogeneity from crude extracts of S. virginiae in five steps, with 5,650-fold purification and 23% overall yield. The N-terminal amino acid sequence was determined to be MAIKLVIA. The purified enzyme showed an apparent Mr of 73,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and an Mr of 280,000 by native molecular sieve high-performance liquid chromatography, indicating the tetrameric nature of the native enzyme. NADPH served as a coenzyme for the reduction, with a Km value of 0.13 mM, but NADH did not support the reaction, even at a concentration of 5 mM, indicating the NADPH-specific nature of the enzyme. The Km for VM1 was determined to be 1.5 mM in the presence of 2 mM NADPH. In the reverse reaction, only 16R-dihydroVM1, not the 16S-epimer, served as a substrate, with a less than 0.1% overall reaction rate compared to that of the forward reaction, confirming that the VM1 reductase participates solely in VM1 inactivation in vivo.     

Identification of N,N'-dicyclohexylcarbodiimide-reactive glutamic and aspartic acid residues in Escherichia coli transhydrogenase and the exchange of these by site-specific mutagenesis

Pyridine nucleotide transhydrogenase (EC 1.6.1.1) from Escherichia coli was investigated with respect to the role of glutamic and aspartic acid residues reactive to N,N'-dicyclohexylcarbodiimide (DCCD) and potentially involved in the proton-pumping mechanism of the enzyme. The E. coli transhydrogenase consists of an alpha (510 residues) and a beta (462 residues) subunit. DCCD reacts with the enzyme to inhibit catalytic activity and proton pumping. This reagent modifies Asp alpha 232, Glu alpha 238, and Glu alpha 240 as well as amino acid residue(s) in the beta subunit. Using the cloned and overexpressed E. coli transhydrogenase genes (Clarke, D. M., and Bragg, P. D. (1985) J. Bacteriol. 162, 367-373), Asp alpha 232 and Glu alpha 238 were replaced independently by site-specific mutagenesis. In addition, Asp alpha 232, Glu alpha 238, and Glu alpha 240 were replaced to generate triple mutants. The specific catalytic activities of the mutant transhydrogenases alpha D232N, alpha D232E, alpha D232K, alpha D232H, alpha E238K, and alpha E238Q as well as of the triple mutants alpha D232N, alpha E238Q, alpha E240Q and alpha D232H, alpha E238Q, alpha E240Q were in the range of 40-90% of the wild-type activity. Proton-pumping activity was present in all mutants. Examination of the extent of subunit modification by [14C]DCCD revealed that the label was still incorporated into both alpha and beta subunits in the Asp alpha 232 mutants, but that the alpha subunit was not labeled in the triple mutants. Catalytic and proton-pumping activities were nearly insensitive to DCCD in the triple mutants. This suggests that loss of catalytic and proton-pumping activities is associated with modification of the aspartic and glutamic acid residues of the alpha subunit. In the presence of the substrate NADPH, the rate of modification of the beta subunit by [14C]DCCD was increased, and there was a greater extent of enzyme inactivation. By contrast, NADH and 3-acetylpyridine-NAD+ protected the catalytic activity of the transhydrogenase from inhibition by DCCD. The protection was particularly marked in the E238Q and E238K mutants. It is concluded that the Asp alpha 232, Glu alpha 238, and Glu alpha 240 residues are not essential for catalytic activity or proton pumping. The inactivation by DCCD is likely due to the introduction of a sterically hindering group that reacts with the identified acidic residues close to the NAD(H)-binding site.     

Monomers of human beta 1 beta 1 alcohol dehydrogenase exhibit activity that differs from the dimer

A previously unreported enzymatic activity is described for monomers of the beta 1 beta 1 isoenzyme of human alcohol dehydrogenase that were prepared from dimeric enzyme by freeze-thaw in liquid nitrogen. Whereas the dimeric enzyme has optimal activity at low substrate concentrations (2.5 mM ethanol, 50 microM NAD+; "low Km" activity), the monomer has its highest activity at high substrate concentrations (1.5 M ethanol, 2.5 mM NAD+; "high Km" activity). While the activity of the monomer does not appear to be saturated at 1.5 M ethanol, its maximal activity at this high ethanol concentration exceeds the Vmax of the dimer by about 3-fold. The apparent Km of NAD+ with monomers is 270 microM, and no activity could be detected with nicotinamide mononucleotide as cofactor. During gel filtration the high Km activity elutes at a lower apparent molecular weight position than the dimer. The kinetics of monomer-to-dimer reassociation are consistent with a second-order process with a rate constant of 240 M-1 s-1. The reassociation rate is markedly enhanced by the presence of NAD+. During refolding of beta 1 beta 1 following denaturation in 6 M guanidine hydrochloride, an enzyme species with high Km activity and spectral properties similar to the freeze-thaw monomer is observed, indicating that a catalytically active monomer is an intermediate in the refolding pathway. The enzymatic activity of the monomer implies that the intersubunit contacts of beta 1 beta 1 are not crucial in establishing a catalytically competent enzyme. However, the differences in specific activity and Km between monomer and dimer suggest that dimerization may serve to modulate the catalytic properties.     

Substrate specificities of catalytic fragments of protein tyrosine phosphatases (HPTP beta, LAR, and CD45) toward phosphotyrosylpeptide substrates and thiophosphotyrosylated peptides as inhibitors

The transmembrane PTPase HPTP beta differs from its related family members in having a single rather than a tandemly duplicated cytosolic catalytic domain. We have expressed the 354-amino acid, 41-kDa human PTP beta catalytic fragment in Escherichia coli, purified it, and assessed catalytic specificity with a series of pY peptides. HPTP beta shows distinctions from the related LAR PTPase and T cell CD45 PTPase domains: it recognizes phosphotyrosyl peptides of 9-11 residues from lck, src, and PLC gamma with Km values of 2, 4, and 1 microM, some 40-200-fold lower than the other two PTPases. With kcat values of 30-205 s-1, the catalytic efficiency, kcat/Km, of the HPTP beta 41-kDa catalytic domain is very high, up to 5.7 x 10(7) M-1 s-1. The peptides corresponding to PLC gamma (766-776) and EGFR (1,167-1,177) phosphorylation sites were used for structural variation to assess pY sequence context recognition by HPTP beta catalytic domain. While exchange of the alanine residue at the +2 position of the PLC gamma (Km of 1 microM) peptide to lysine or aspartic acid showed little or no effect on substrate affinity, replacement by arginine increased the Km 35-fold. Similarly, the high Km value of the EGFR pY peptide (Km of 104 microM) derives largely from the arginine residue at the +2 position of the peptide, since arginine to alanine single mutation at the -2 position of the EGFR peptide decreased the Km value 34-fold to 3 microM. Three thiophosphotyrosyl peptides have been prepared and act as substrates and competitive inhibitors of these PTPase catalytic domains.