Chemical modification of 3 alpha,20 beta-hydroxysteroid dehydrogenase with diethyl pyrocarbonate. Evidence for an essential, highly reactive, lysyl residue

Diethyl pyrocarbonate inactivated the tetrameric 3 alpha,20 beta-hydroxysteroid dehydrogenase with second-order rate constants of 1.63 M-1 s-1 at pH 6 and 25 degrees C or 190 M-1 s-1 at pH 9.4 and 25 degrees C. The activity was slowly and partially restored by incubation with hydroxylamine (81% reactivation after 28 h with 0.1 M hydroxylamine, pH 9, 25 degrees C). NADH protected the enzyme against inactivation with a Kd (10 microM) very close to the Km (7 microM) for the coenzyme. The ultraviolet difference spectrum of inactivated vs. native enzyme indicated that a single histidyl residue per enzyme subunit was modified by diethyl pyrocarbonate, with a second-order rate constant of 1.8 M-1 s-1 at pH 6 and 25 degrees C. The histidyl residue, however, was not essential for activity because in the presence of NADH it was modified without enzyme inactivation and modification of inactivated enzyme was rapidly reversed by hydroxylamine without concomitant reactivation. Progesterone, in the presence of NAD+, protected the histidyl residue against modification, and this suggests that the residue is located in or near the steroid binding site of the enzyme. Diethyl pyrocarbonate also modified, with unusually high reaction rate, one lysyl residue per enzyme subunit, as demonstrated by dinitrophenylation experiments carried out on the treated enzyme. The correlation between inactivation and modification of lysyl residues at different pHs and the protection by NADH against both inactivation and modification of lysyl residues indicate that this residue is essential for activity and is located in or near the NADH binding site of the enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)     

Reversible inactivation of Saccharomyces cerevisiae glutathione reductase under reducing conditions

Glutathione reductase from Saccharomyces cerevisiae was rapidly inactivated following aerobic incubation with NADPH, NADH, and several other reductants, in a time- and temperature-dependent process. The inactivation had already reached 50% when the NADPH concentration reached that of the glutathione reductase subunit. The inactivation was very marked at pH values below 5.5 and over 7, while only a slight activity decrease was noticed at pH values between these two values. After elimination of excess NADPH the enzyme remained inactive for at least 4 h. The enzyme was protected against redox inactivation by low concentrations of GSSG, ferricyanide, GSH, or dithiothreitol, and high concentrations of NAD(P)+; oxidized glutathione effectively protected the enzyme at concentrations even lower than GSH. The inactive enzyme was efficiently reactivated after incubation with GSSG, ferricyanide, GSH, or dithiothreitol, whether NADPH was present or not. The reactivation with GSH was rapid even at 0 degree C, whereas the optimum temperature for reactivation with GSSG was 30 degrees C. A tentative model for the redox interconversion, involving an erroneous intramolecular disulfide bridge, is put forward.     

Multiparameter study of denaturation of 20 beta-hydroxysteroid dehydrogenase by urea in the presence of stabilizing agents

We investigated the denaturation of tetrameric 20 beta-hydroxysteroid dehydrogenase (20R)-17 beta,20 beta,21-trihydroxysteroid:NAD+ oxidoreductase, EC 1.1.1.53) to find out whether intermediate states are formed during the process. The denaturation process was studied in the presence and absence of stabilizers, both specific, such as NADH, and non-specific, such as the salting-out anion phosphate. Changes in enzymatic activity, intrinsic protein fluorescence and far-ultraviolet circular dichroism were monitored. When NADH was present, denaturation of the enzyme by urea was a one-step transition between the native and the completely denatured state. In dilute phosphate, and even more so in concentrated phosphate, the existence of intermediate states with different stability is evidenced by the noncoincidence of the transition curves that probe for different functional and conformational aspects of the enzyme. Therefore, for 20 beta-hydroxysteroid dehydrogenase the formation of intermediates can be prevented by adding NADH, or enhanced by adding concentrated phosphate.     

Structural and catalytic properties of L-alanine dehydrogenase from Bacillus cereus

Alanine dehydrogenase from Bacillus cereus, a non-allosteric enzyme composed of six identical subunits, was purified to homogeneity by chromatography on blue-Sepharose and Sepharose 6B-CL. Like other pyridine-linked dehydrogenases, alanine dehydrogenase is inhibited by Cibacron blue, competitively with respect to NADH and noncompetitively with respect to pyruvate. The enzyme was inactivated by 0.1 M glycine/HCl (pH 2) and reactivated by 0.1 M phosphate (pH 8) supplemented with NAD+ or NADH. The reactivation was characterized by sigmoidal kinetics indicating a complex mechanism involving rate-limiting folding and association steps. Cibacron blue interfered with renaturation, presumably by competition with NADH. Chromatography on Sepharose 6B-CL of the partially renatured alanine dehydrogenase led to the separation of several intermediates, but only the hexamer was characterized by enzymatic activity. By immobilization on Sepharose 4B, alanine dehydrogenase from B. cereus retained 66% of the specific activity of the soluble enzyme. After denaturation of immobilized alanine dehydrogenase with 7 M urea, 37% of the initial protein was still bound to Sepharose, indicating that on the average the hexamer was attached to the matrix via, at most, two subunits. The ability of the denatured, immobilized subunits to pick up subunits from solution shows their capacity to fold back to the native conformation after urea treatment. The formation of "hybrids" between subunits of enzyme from B. cereus and Bacillus subtilis demonstrates the close resemblance of the tertiary and quaternary structures of alanine dehydrogenases from these species.     

Reversible modification of D-beta-hydroxybutyrate dehydrogenase by diamide

D-beta-Hydroxybutyrate dehydrogenase is a lipid-requiring enzyme with a specific requirement of lecithin for function. The purified enzyme devoid of lipid (apodehydrogenase) is inactive but can be reactivated by forming a complex with phospholipid containing lecithin. We find that, of the six half cysteines present in D-beta-hydroxybutyrate dehydrogenase, only two are in the reduced form and available for modification with N-ethylmaleimide, even after denaturation in sodium dodecyl sulfate. Diamide treatment of either the inactive apodehydrogenase or the active enzyme-phospholipid complex resulted in complete loss of enzymic activity, the apodehydrogenase being assayed after addition of phospholipid. The inactivation by diamide can be reversed by the addition of dithiothreitol with full recovery of activity. Derivatization using N-[14C]ethylmaleimide showed that diamide modified only one sulfhydryl per enzyme monomer. The other sulfhydryl appears not to be essential for function since full activity can be restored after this sulfhydryl had been covalently derivatized with N-ethylmaleimide. Protein cross-linking was not observed after diamide modification of D-beta-hydroxybutyrate dehydrogenase, indicating that a disulfide bridge was not formed between enzyme subunits. The diamide-modified enzyme retains the ability to bind coenzyme, NAD(H), as detected by quenching of the intrinsic fluorescence of the protein. However, resonance energy transfer from protein to bound NADH and enhancement of NADH fluorescence were not observed, indicating that diamide modification of the protein alters the nucleotide binding site.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of the lyotropic series of anions on denaturation and renaturation of 20 beta-hydroxysteroid dehydrogenase

The effect of the lyotropic series of anions on the stability and renaturation of tetrameric 20 beta-hydroxysteroid dehydrogenase (17,20 beta,21-trihydroxysteroid:NAD+ oxidoreductase, EC 1.1.1.53) was investigated. The variations in enzymatic activity were correlated with the changes in protein fluorescence, circular dichroism, reactivity of histidine residues and molecular weight. High concentrations of salting-out anions (phosphate, citrate, sulphate) were found to stabilize the enzyme markedly and increase the renaturation yield of the urea-denatured enzyme. Phosphate, for instance, induced the highest stabilization at about 1.2 M and the maximum reactivation (66%) at 0.5 M. At low anion concentration (0.01 M), the reactivation was only 7%. The renaturation property of salting-out anions seems to be due to their stabilizing effect on the end-product, i.e., the assembled tetramer. Salting-in anions (perchlorate, thiocyanate, iodide) inactivated the enzyme. At moderate anion concentrations (no greater than 0.25 M) the activation, which occurred slowly, without tetramer dissociation and with minor modifications of enzyme conformation, was fully reversed by concentrated phosphate or by saturating concentrations of NADH. In contrast, the inactivation induced by high anion concentrations (1-2 M) was rapid, irreversible and linked to considerable modifications of enzyme conformation.     

Reactivity of the essential thiol group of lactate dehydrogenase and substrate binding

1. The preparation of a derivative of pig heart lactate dehydrogenase in which the essential thiol group has been converted into an S-sulpho group is described. The derivative has unchanged s(20,w) and is catalytically inactive. 2. The rate of reaction of the essential thiol group is controlled by a system with a pK>9. 3. The essential thiol group is protected by NADH against reaction with maleimide. 4. Lactate dehydrogenase in which the essential thiol group has been converted into an S-sulpho group or alkylated with maleimide still binds one molecule of NADH/subunit but with a three- to four-fold diminished affinity. 5. The inhibited enzymes also bind one molecule of NAD(+)-sulphite complex/subunit but with affinity decreased 10(3)-10(4)-fold. 6. The inhibited enzymes fail to bind C(2) and C(3) molecules to give the ternary complexes enzyme-NAD(+)-pyruvate, enzyme-NADH-oxamate and enzyme-NADH-oxalate. The 1:1:1 stoicheiometry of the last-mentioned complex with the native enzyme was established by gel filtration. 7. Structures that account for these results are discussed.     

Testicular and adrenal 3 beta-hydroxy-5-ene-steroid dehydrogenase and 5-ene-4-ene isomerase

The purified multifunctional enzyme, 3 beta-hydroxysteroid dehydrogenase with steroid 5-ene-4-ene isomerase from rat testes and adrenals showed similar catalytic properties. They exhibited the same molecular weight of 46,500. Either NAD+ or NADH was required for steroid isomerizing activity, probably as an allosteric effector. It was clearly demonstrated by using the purified enzyme that without NAD(H) no isomerizing activity was detected. In the presence of NADH, or its analogue, 3 beta-hydroxysteroid dehydrogenase obtained from both tissues was inhibited; however, steroid isomerizing activity remained due to the allosteric effect. The results suggest that in these endocrine organs, both enzyme activities reside within the same protein.     

Enzymatic properties, renaturation and metabolic role of mannitol-1-phosphate dehydrogenase from Escherichia coli

Enzymatic properties, renaturation and metabolic role of mannitol-1-phosphate dehydrogenase from Escherichia coli. D-mannitol-1-phosphate dehydrogenase was purified to homogeneity from Escherichia coli, and its physicochemical and enzymatic properties were investigated. The molecular weight of the polypeptide chain is 45,000 as determined by polyacrylamide gel electrophoresis in denaturing conditions. High performance size exclusion chromatography gives an apparent molecular weight of 47,000 for the native enzyme, showing that D-mannitol-1-phosphate dehydrogenase is a monomeric NAD-dependent dehydrogenase. D-mannitol-1-phosphate dehydrogenase is rapidly denatured by 6 M guanidine hydrochloride. Non-superimposable transition curves for the loss of activity and the changes in fluorescence suggest the existence of a partially folded inactive intermediate. The protein can be fully renatured after complete unfolding, and the regain of both native fluorescence and activity occurs rapidly within a few seconds at pH 7.5 and 20 degrees C. Such a high rate of reactivation is unusual for a protein of this size. D-mannitol-1-phosphate dehydrogenase is specific for mannitol-1-phosphate (or fructose-6-phosphate) as a substrate and NAD+ (or NADH) as a cofactor. Zinc is not required for the activity. The affinity of D-mannitol-1-phosphate dehydrogenase for the reduced or oxidized form of its substrate or cofactor remains constant with pH. The affinity for NADH is 20-fold higher than for NAD+. The forward and reverse catalytic rate constants of the reaction: mannitol-1-phosphate + NAD+ in equilibrium fructose-6-phosphate + NADH have different pH dependences. The oxidation of mannitol-1-phosphate has an optimum pH of 9.5, while the reduction of fructose-6-phosphate has its maximum rate at pH 7.0. At pH values around neutrality the maximum rate of reduction of fructose-6-phosphate is much higher than that of oxidation of mannitol-1-phosphate. The enzymatic properties of isolated D-mannitol-1-phosphate dehydrogenase are discussed in relation to the role of this enzyme in the intracellular metabolism.     

Inhibition of the reactivation of acid-dissociated lactate dehydrogenase isoenzymes by their aminoterminal CNBr fragments

The catalytic activity of lactate dehydrogenase isoenzymes (LDH) depends on their tetrameric structure. Stabilization of this quaternary structure is achieved by interaction of the N-terminal part of one subunit with the C-terminal region of the other subunit. The N-terminal peptides from pig M-LDH and H-LDH which are responsible for this stabilization were obtained by CNBr-fragmentation and purification on reversed-phase HPLC. The effect of these peptides on the formation of the quaternary structure of LDH-isoenzymes was investigated by monitoring the reconstitution of the catalytic activity after acid-dissociation. Low concentrations of the N-terminal peptides led to an increased, and high concentrations to a decreased yield of reconstituted LDH activity. The effects of these two peptides were isoenzyme specific. The 32 residue peptide derived from M-LDH showed the highest effect when tested with M-LDH as target enzyme but only a poor effect with H-LDH. On the other side the 33 residue peptide generated from H-LDH showed a moderate effect with both isoenzymes. The effects of the N-terminal LDH peptides are antagonized by the coenzymes NAD+ and NADH. The most significant influence was observed with NAD+ in the M-LDH peptide-M-LDH enzyme system. Comparison of the properties of the reactivation antagonists isolated from human origin with the N-terminal CNBr-peptides of LDH revealed identity in all essential properties, suggesting that the former peptides are generated by degradation of LDH.     

Reversible Inactivation of Nitrate Reductase by NADH and the Occurrence of Partially Inactive Enzyme in the Wheat Leaf

Nitrate reductase from wheat (Triticum aestivum L. cv Bindawarra) leaves is inactivated by pretreatment with NADH, in the absence of nitrate, a 50% loss of activity occurring in 30 minutes at 25°C with 10 micromolar NADH. Nitrate (50 micromolar) prevented inactivation by 10 micromolar NADH while cyanide (1 micromolar) markedly enhanced the degree of inactivation.

A rapid reactivation of NADH-inactivated nitrate reductase occurred after treatment with 0.3 millimolar ferricyanide or exposure to light (230 milliwatts per square centimeter) plus 20 micromolar flavin adenine dinucleotide. When excess NADH was removed, the enzyme was also reactivated by autoxidation. Nitrate did not influence the rate of reactivation.

Leaf nitrate reductase, from plants grown for 12 days on 1 millimolar nitrate, isolated in the late photoperiod or dark period, was activated by ferricyanide or light treatment. This suggests that, at these times of the day, the nitrate reductase in the leaves of the low nitrate plants is in a partially inactive state (NADH-inactivated). The nitrate reductase from moisture-stressed plants showed a greater degree of activation after light treatment, and inactive enzyme in them was detected earlier in the photoperiod.

     

Purification, reconstitution, and steady-state kinetics of the trans-membrane 17 beta-hydroxysteroid dehydrogenase 2

Human membrane 17 beta-hydroxysteroid dehydrogenase 2 is an enzyme essential in the conversion of the highly active 17beta-hydroxysteroids into their inactive keto forms in a variety of tissues. 17 beta-hydroxysteroid dehydrogenase 2 with 6 consecutive histidines at its N terminus was expressed in Sf9 insect cells. This recombinant protein retained its biological activity and facilitated the enzyme purification and provided the most suitable form in our studies. Dodecyl-beta-D-maltoside was found to be the best detergent for the solubilization, purification, and reconstitution of this enzyme. The overexpressed integral membrane protein was purified with a high catalytic activity and a purity of more than 90% by nickel-chelated chromatography. For reconstitution, the purified protein was incorporated into dodecyl-beta-D-maltoside-destabilized liposomes prepared from l-alpha-phosphatidylcholine. The detergent was removed by adsorption onto polystyrene beads. The reconstituted enzyme had much higher stability and catalytic activity (2.6 micromol/min/mg of enzyme protein with estradiol) than the detergent-solubilized and purified protein (0.9 micromol/min/mg of enzyme protein with estradiol). The purified and reconstituted protein (with a 2-kDa His tag) was proved to be a homodimer, and its functional molecular mass was calculated to be 90.4 +/- 1.2 kDa based on glycerol gradient analytical ultracentrifugation and chemical cross-linking study. The kinetic studies demonstrated that 17 beta-hydroxysteroid dehydrogenase 2 was an NAD-preferring dehydrogenase with the K(m) of NAD being 110 +/- 10 microM and that of NADP 9600 +/- 100 microM using estradiol as substrate. The kinetic constants using estradiol, testosterone, dihydrotestosterone, and 20 alpha-dihydroprogesterone as substrates were also determined.     

UDP-galactose 4-epimerase from Escherichia coli: equilibrium unfolding studies

UDP-galactose 4-epimerase from Escherichia coli is a homodimer of 39 kDa subunit with non-covalently bound NAD acting as cofactor. The enzyme can be reversibly reactivated after denaturation and dissociation using 8 M urea at pH 7.0. There is a strong affinity between the cofactor and the refolded molecule as no extraneous NAD is required for its reactivation. Results from equilibrium denaturation using parameters like catalytic activity, circular-dichroism, fluorescence emission (both intrinsic and with extraneous fluorophore 1-aniline 8-naphthalene sulphonic acid), 'reductive inhibition' (associated with orientation of NAD on the native enzyme surface), elution profile from size-exclusion HPLC and light scattering have been compiled here. These show that inactivation, integrity of secondary, tertiary and quaternary structures have different transition mid-points suggestive of non-cooperative transition. The unfolding process may be broadly resolved into three parts: an active dimeric holoenzyme with 50% of its original secondary structure at 2.5 M urea; an active monomeric holoenzyme at 3 M urea with only 40% of secondary structure and finally further denaturation by 6 M urea leads to an inactive equilibrium unfolded state with only 20% of residual secondary structure. Thermodynamical parameters associated with some transitions have been quantitated. The results have been discussed with the X-ray crystallographic structure of the enzyme.     

Horse liver alcohol dehydrogenase. A study of the essential lysine residue.

1. The inactivation of horse liver alcohol dehydrogenase by pyridoxal 5'-phosphate in phosphate buffer, pH8, at 10 degrees C was investigated. Activity declines to a minimum value determined by the pyridoxal 5'-phosphate concentration. The maximum inactivation in a single treatment is 75%. This limit appears to be set by the ratio of the first-order rate constants for interconversion of inactive covalently modified enzyme and a readily dissociable non-covalent enzyme-modifier complex. 2. Reactivation was virtually complete on 150-fold dilution: first-order analysis yielded an estimate of the rate constant (0.164min-1), which was then used in the kinetic analysis of the forward inactivation reaction. This provided estimates for the rate constant for conversion of non-covalent complex into inactive enzyme (0.465 min-1) and the dissociation constant of the non-covalent complex (2.8 mM). From the two first-order constants, the minimum attainable activity in a single cycle of treatment may be calculated as 24.5%, very close to the observed value. 3. Successive cycles of modification followed by reduction with NaBH4 each decreased activity by the same fraction, so that three cycles with 3.6 mM-pyridoxal 5'-phosphate decreased specific activity to about 1% of the original value. The absorption spectrum of the enzyme thus treated indicated incorporation of 2-3 mol of pyridoxal 5'-phosphate per mol of subunit, covalently bonded to lysine residues. 4. NAD+ and NADH protected the enzyme completely against inactivation by pyridoxal 5'-phosphate, but ethanol and acetaldehyde were without effect. 5. Pyridoxal 5'-phosphate used as an inhibitor in steady-state experiments, rather than as an inactivator, was non-competitive with respect to both NADH and acetaldehyde. 6. The partially modified enzyme (74% inactive) showed unaltered apparent Km values for NAD+ and ethanol, indicating that modified enzyme is completely inactive, and that the residual activity is due to enzyme that has not been covalently modified. 7. Activation by methylation with formaldehyde was confirmed, but this treatment does not prevent subsequent inactivation with pyridoxal 5'-phosphate. Presumably different lysine residues are involved. 8. It is likely that the essential lysine residue modified by pyridoxal 5'-phosphate is involved either in binding the coenzymes or in the catalytic step. 9. Less detailed studies of yeast alcohol dehydrogenase suggest that this enzyme also possesses an essential lysine residue.     

Purification and characterization of rat adrenal 3 beta-hydroxysteroid dehydrogenase with steroid 5-ene-4-ene-isomerase

After solubilization of rat adrenal microsomes with sodium cholate, 3 beta-hydroxysteroid dehydrogenase with steroid 5-ene-4-ene isomerase (abbreviated as steroid isomerase) activity was purified to a homogeneous state. The following characteristics of the enzyme were obtained: 3 beta-Hydroxysteroid dehydrogenase together with steroid isomerase was detected as a single protein band in SDS-polyacrylamide gel electrophoresis, where its mol. wt was estimated as 46,500. Either NAD+ or NADH was required for demonstration of steroid isomerase activity. Treatment of the enzyme with 5'-p-fluorosulfonylbenzoyladenosine, an affinity labeling reagent for NAD+-dependent enzyme, diminished both the enzyme activities.     

Purification and characterization of 17 beta-hydroxysteroid dehydrogenase from Cylindrocarpon radicicola

An NAD+-linked 17 beta-hydroxysteroid dehydrogenase was purified to homogeneity from a fungus, Cylindrocarpon radicicola ATCC 11011 by ion exchange, gel filtration, and hydrophobic chromatographies. The purified preparation of the dehydrogenase showed an apparent molecular weight of 58,600 by gel filtration and polyacrylamide gel electrophoresis. SDS-gel electrophoresis gave Mr = 26,000 for the identical subunits of the protein. The amino-terminal residue of the enzyme protein was determined to be glycine. The enzyme catalyzed the oxidation of 17 beta-hydroxysteroids to the ketosteroids with the reduction of NAD+, which was a specific hydrogen acceptor, and also catalyzed the reduction of 17-ketosteroids with the consumption of NADH. The optimum pH of the dehydrogenase reaction was 10 and that of the reductase reaction was 7.0. The enzyme had a high specific activity for the oxidation of testosterone (Vmax = 85 mumol/min/mg; Km for the steroid = 9.5 microM; Km for NAD+ = 198 microM at pH 10.0) and for the reduction of androstenedione (Vmax = 1.8 mumol/min/mg; Km for the steroid = 24 microM; Km for NADH = 6.8 microM at pH 7.0). In the purified enzyme preparation, no activity of 3 alpha-hydroxysteroid dehydrogenase, 3 beta-hydroxysteroid dehydrogenase, delta 5-3-ketosteroid-4,5-isomerase, or steroid ring A-delta-dehydrogenase was detected. Among several steroids tested, only 17 beta-hydroxysteroids such as testosterone, estradiol-17 beta, and 11 beta-hydroxytestosterone, were oxidized, indicating that the enzyme has a high specificity for the substrate steroid. The stereospecificity of hydrogen transfer by the enzyme in dehydrogenation was examined with [17 alpha-3H]testosterone.     

Redox interconversion of Escherichia coli glutathione reductase. A study with permeabilized and intact cells

Summary The redox interconversion of Escherichia coli glutathione reductase has been studied both in situ, with permeabilized cells treated with different reductants, and in vivo, with intact cells incubated with compounds known to alter their intracellular redox state.The enzyme from toulene-permeabilized cells was inactivated in situ by NADPH, NADH, dithionite, dithiothreitol, or GSH. The enzyme remained, however, fully active upon incubation with the oxidized forms of such compounds. The inactivation was time-, temperature-, and concentration-dependent; a 50% inactivation was promoted by just 2 M NADPH, while 700 M NADH was required for a similar effect. The enzyme from permeabilized cells was completely protected against redox inactivation by GSSG, and to a lesser extent by dithiothreitol, GSH, and NAD(P)⁺. The inactive enzyme was efficiently reactivated in situ by physiological GSSG concentrations. A significant reactivation was promoted also by GSH, although at concentrations two orders of magnitude below its physiological concentrations. The glutathione reductase from intact E. coli cells was inactivated in vivo by incubation with DL-malate, DL-isocitrate, or higher L-lactate concentrations. The enzyme was protected against redox inactivation and fully reactivated by diamide in a concentration-dependent fashion. Diamide reactivation was not dependent on the synthesis of new protein, thus suggesting that the effect was really a true reactivation and not due to de novo synthesis of active enzyme. The glutathione reductase activity increased significantly after incubation of intact cells with tert-butyl or cumene hydroperoxides, suggesting that the enzyme was partially inactive within such cells. In conclusion, the above results show that both in situ and in vivo the glutathione reductase of Escherichia coli is subjected to a redox interconversion mechanism probably controlled by the intracellular NADPH and GSSG concentrations.     

The binding of oxidized and reduced nicotinamide–adenine dinucleotides to bovine liver uridine diphosphate glucose dehydrogenase

The binding of NAD(+) and NADH to bovine liver UDP-glucose dehydrogenase was studied by using gel-filtration and fluorescence-titration methods. The enzyme bound 0.5mol of NAD(+) and 2 mol of NADH/mol of subunit at saturating concentrations of both substrate and product. The dissociation constant for NADH was 4.3mum. The binding of NAD(+) to the enzyme resulted in a small quench of protein fluorescence whereas the binding of NADH resulted in a much larger (60-70%) quench of protein fluorescence. The binding of NADH to the enzyme was pH-dependent. At pH8.1 a biphasic profile was obtained on titrating the enzyme with NADH, whereas at pH8.8 the titration profile was hyperbolic. UDP-xylose, and to a lesser extent UDP-glucuronic acid, lowered the apparent affinity of the enzyme for NADH.     

Isolation and characterisation of the glycerol dehydrogenase from Bacillus stearothermophilus

A protocol for the rapid purification of the glycerol dehydrogenase (glycerol: NAD+ 2-oxidoreductase, EC 1.1.1.6) from the thermophile Bacillus stearothermophilus has been developed using a combination of chromatographic techniques including affinity chromatography on a Sepharose-immobilised triazine dye (Procion red, HE3B, ICI). Substrate specificity has been examined and Km values determined. The protein has been shown to have an oligomeric Mr of approx. 180,000 and consists of four identical subunits of Mr 42,000. Exposure to chelating agents (e.g., EDTA) leads to total loss of activity; the EDTA-inactivated enzyme can be reactivated by Zn2+ and requires 1 mol equivalent of zinc per subunit for full catalytic activity. Other divalent cations such as Cd2+ and Co2+ will reactivate the apo-enzyme but yields an enzyme of lower specific activity. The enzyme binds 1 equivalent of NADH per subunit and during catalysis transfers the 4-pro-R hydride from the nicotinamide ring of the reduced-coenzyme to the substrate. Glycerol increases the dissociation constant for the interaction between NADH and Zn-metallo-glycerol dehydrogenase (ZnGDH) but has no effect on the equilibrium between NADH and metal-depleted enzyme.     

Inactivation of Escherichia coli L-threonine dehydrogenase by 2,3-butanedione. Evidence for a catalytically essential arginine residue

Incubation of homogeneous preparations of L-threonine dehydrogenase from Escherichia coli with 2,3-butanedione, 2,3-pentanedione, phenylglyoxal, or 1,2-cyclohexanedione causes a time- and concentration-dependent loss of enzymatic activity; plots of log percent activity remaining versus time are linear to greater than 90% inactivation, indicative of pseudo-first order inactivation kinetics. The reaction order with respect to the concentration of modifying reagent is approximately 1.0 in each case suggesting that the loss of catalytic activity is due to one molecule of modifier reacting with each active unit of enzyme. Controls establish that this inactivation is not due to modifier-induced dissociation or photoinduced nonspecific alteration of the dehydrogenase. Essentially the same Km but decreased Vmax values are obtained when partially inactivated enzyme is compared with native. NADH (25 mM) and NAD+ (70 mM) give full protection against inactivation whereas much higher concentrations (i.e. 150 mM) of L-threonine or L-threonine amide provide a maximum of 80-85% protection. Amino acid analyses coupled with quantitative sulfhydryl group determinations show that enzyme inactivated 95% by 2,3-butanedione loses 7.5 arginine residues (out of 16 total)/enzyme subunit with no significant change in other amino acid residues. In contrast, only 2.4 arginine residues/subunit are modified in the presence of 80 mM NAD+. Analysis of the course of modification and inactivation by the statistical method of Tsou (Tsou, C.-L. (1962) Sci. Sin. 11, 1535-1558) demonstrates that inactivation of threonine dehydrogenase correlates with the loss of 1 "essential" arginine residue/subunit which quite likely is located in the NAD+/NADH binding site.