Enzymic and chemical synthesis of epilson-N-(L-propionyl-2)-L-lysine.

Pyruvate was shown to act as an oxo acid substrate in the reverse direction of saccharopine dehydrogenase [epsilon N-(L-glutaryl-2)-L-lysine: NAD oxidoreductase (L-lysine-forming)] reaction. The enzymic condensation product of lysine and pyruvate was isolated and identified as epsilon-N-(L-propionyl-2)-L-lysine by comparison with the synthetic compound. A method for the chemical preparation of diastereoisomers of epsilon-N-(propionyl-2)-L-lysine is also described.     

Saccharopine dehydrogenase. Substrate inhibition studies.

In the direction of reductive condensation of alpha-ketoglutarate and lysine, saccharopine dehydrogenase (N6-(glutar-2-yl)-L-lysine:NAD oxidoreductase (lysine-forming) is inhibited by high concentrations of alpha-ketoglutarate and lysine, but not by NADH. NAD+ and saccharopine show no substrate inhibition in the reverse direction. Substrate inhibition by alpha-ketoglutarate and lysine is linear uncompetitive versus NADH. However, when the inhibition is examined with alpha-ketoglutarate or lysine as the variable substrate, the double reciprocal plots show a family of curved lines concave up. The curvature is more pronounced with increasing concentrations of the inhibitory substrate, suggesting an interaction of variable substrate with the enzyme form carrying the inhibitory substrate. These inhibition patterns, the lack of interaction of structural analogs of lysine such as ornithine and norleucine with the E-NAD+ complex (Fujioka M., and Nakatani, Y. (1972) Eur. J. Biochem. 25, 301-307), the identity of values of inhibition constants of alpha-ketoglutarate and lysine obtained with either one as the substrate inhibitor, and the substrate inhibition data in the presence of a reaction product, NAD+, are consistent with the mechanism that substrate inhibition results from the formation of a dead-end E-NAD+-alpha-ketoglutarate complex followed by the addition of lysine to this abortive complex.     

Overall kinetic mechanism of saccharopine dehydrogenase from Saccharomyces cerevisiae

Kinetic data have been measured for the histidine-tagged saccharopine dehydrogenase from Saccharomyces cerevisiae, suggesting the ordered addition of nicotinamide adenine dinucleotide (NAD) followed by saccharopine in the physiologic reaction direction. In the opposite direction, the reduced nicotinamide adenine dinucleotide (NADH) adds to the enzyme first, while there is no preference for the order of binding of alpha-ketoglutarate (alpha-Kg) and lysine. In the direction of saccharopine formation, data also suggest that, at high concentrations, lysine inhibits the reaction by binding to free enzyme. In addition, uncompetitive substrate inhibition by alpha-Kg and double inhibition by NAD and alpha-Kg suggest the existence of an abortive E:NAD:alpha-Kg complex. Product inhibition by saccharopine is uncompetitive versus NADH, suggesting a practical irreversibility of the reaction at pH 7.0 in agreement with the overall K(eq). Saccharopine is noncompetitive versus lysine or alpha-Kg, suggesting the existence of both E:NADH:saccharopine and E:NAD:saccharopine complexes. NAD is competitive versus NADH, and noncompetitive versus lysine and alpha-Kg, indicating the combination of the dinucleotides with free enzyme. Dead-end inhibition studies are also consistent with the random addition of alpha-Kg and lysine. Leucine and oxalylglycine serve as lysine and alpha-Kg dead-end analogues, respectively, and are uncompetitive against NADH and noncompetitive against alpha-Kg and lysine, respectively. Oxaloacetate (OAA), pyruvate, and glutarate behave as dead-end analogues of lysine, which suggests that the lysine-binding site has a higher affinity for keto acid analogues than does the alpha-Kg site or that dicarboxylic acids have more than one binding mode on the enzyme. In addition, OAA and glutarate also bind to free enzyme as does lysine at high concentrations. Glutarate gives S-parabolic noncompetitive inhibition versus NADH, indicating the formation of a E:(glutarate)2 complex as a result of occupying both the lysine- and alpha-Kg-binding sites. Pyruvate, a slow alternative keto acid substrate, exhibits competitive inhibition versus both lysine and alpha-Kg, suggesting the combination to the E:NADH:alpha-Kg and E:NADH:lysine enzyme forms. The equilibrium constant for the reaction has been measured at pH 7.0 as 3.9 x 10(-7) M by monitoring the change in NADH upon the addition of the enzyme. The Haldane relationship is in very good agreement with the directly measured value.     

A kinetic study of the alpha-keto acid dehydrogenase complexes from pig heart mitochondria.

The kinetic mechanisms of the 2-oxoglutarate and pyruvate dehydrogenease complexes from pig heart mitochondria were studied at pH 7.5 and 25 degrees. A three-site ping-pong mechanism for the actin of both complexes was proposed on the basis of the parallel lines obtained when 1/v was plotted against 2-oxoglutarate or pyruvate concentration for various levels of CoA and a level of NAD+ near its Michaelis constant value. Rate equations were derived from the proposed mechanism. Michaelis constants for the reactants of the 2-oxoglutarate dehydrogenase complex reaction are: 2-oxoglutarate, 0.220 mM; CoA, 0.025 mM; NAD+, 0.050 mM. Those of the pyruvate dehydrogenase complex are: pyruvate, 0.015 mM; CoA, 0.021 mM; NAD+, 0.079 mM. Product inhibition studies showed that succinyl-CoA or acetyl-CoA was competitive with respect to CoA, and NADH was competitive with respect to NAD+ in both overall reactions, and that succinyl-CoA or acetyl-CoA and NADH were uncompetitive with respect to 2-oxoglutarate or pyruvate, respectively. However, noncompetitive (rather than uncompetitive) inhibition patterns were observed for succinyl-CoA or acetyl-CoA versus NAD+ and for NADH versus CoA. These results are consistent with the proposed mechanisms.     

Purification and properties of tauropine dehydrogenase from the shell adductor muscle of the ormer, Haliotis lamellosa

Tauropine dehydrogenase (tauropine:NAD oxidoreductase) was purified from the shell adductor muscle of the ormer, Haliotis lamellosa. The enzyme was found to utilize stoichiometrically NADH as co-enzyme and pyruvate and taurine as substrates producing tauropine [rhodoic acid; N-(D-1-carboxyethyl)-taurine]. The enzyme was purified to a specific activity of 463 units/mg protein using a combination of ammonium sulphate fractionation, ion-exchange and affinity chromatography. The relative molecular mass was 38,000 +/- 1000 when assessed by gel filtration on Ultrogel AcA 54 and 42,000 +/- 150 by electrophoresis on 5-10% polyacrylamide gels in the presence of 1% sodium dodecyl sulphate; the data suggest a monomeric structure. Tauropine and pyruvate were found to be the preferred substrates. Among the amino acids tested for activity with the enzyme, only alanine is used as an alternative substrate, but with a rate less than 6% of the enzyme activity with taurine. Of the oxo acids tested, 2-oxobutyrate and 2-oxovalerate were also found to be substrates. Apparent Km values for the substrates NADH, pyruvate and taurine are 0.022 +/- 0.003 mM, 0.64 +/- 0.07 mM and 64.7 +/- 5.4 mM, respectively, at pH 7.0 and for the products, NAD+ and tauropine, are 0.29 +/- 0.01 mM and 9.04 +/- 1.27 mM, respectively, at pH 8.3. Apparent Km values for both pyruvate and taurine decrease with increasing co-substrate (taurine or pyruvate) concentration. NAD+ and tauropine were found to be product inhibitors of the forward reaction. NAD+ was a competitive inhibitor of NADH, whereas tauropine gave a mixed type of inhibition with respect to pyruvate and taurine. Succinate was found to inhibit non-competitively with respect to taurine and pyruvate with an apparent Ki value in the physiological range of this anaerobic end product. The inhibition by L-lactate, not an end product in the ormer, was competitive with respect to pyruvate. The physiological role or tauropine dehydrogenase during anaerobiosis is discussed.     

A proposed proton shuttle mechanism for saccharopine dehydrogenase from Saccharomyces cerevisiae

Saccharopine dehydrogenase [N6-(glutaryl-2)-L-lysine:NAD oxidoreductase (L-lysine forming)] catalyzes the final step in the alpha-aminoadipate pathway for lysine biosynthesis. It catalyzes the reversible pyridine nucleotide-dependent oxidative deamination of saccharopine to generate alpha-Kg and lysine using NAD+ as an oxidizing agent. The proton shuttle chemical mechanism is proposed on the basis of the pH dependence of kinetic parameters, dissociation constants for competitive inhibitors, and isotope effects. In the direction of lysine formation, once NAD+ and saccharopine bind, a group with a pKa of 6.2 accepts a proton from the secondary amine of saccharopine as it is oxidized. This protonated general base then does not participate in the reaction again until lysine is formed at the completion of the reaction. A general base with a pKa of 7.2 accepts a proton from H2O as it attacks the Schiff base carbon of saccharopine to form the carbinolamine intermediate. The same residue then serves as a general acid and donates a proton to the carbinolamine nitrogen to give the protonated carbinolamine. Collapse of the carbinolamine is then facilitated by the same group accepting a proton from the carbinolamine hydroxyl to generate alpha-Kg and lysine. The amine nitrogen is then protonated by the group that originally accepted a proton from the secondary amine of saccharopine, and products are released. In the reverse reaction direction, finite primary deuterium kinetic isotope effects were observed for all parameters with the exception of V2/K(NADH), consistent with a steady-state random mechanism and indicative of a contribution from hydride transfer to rate limitation. The pH dependence, as determined from the primary isotope effect on DV2 and D(V2/K(Lys)), suggests that a step other than hydride transfer becomes rate-limiting as the pH is increased. This step is likely protonation/deprotonation of the carbinolamine nitrogen formed as an intermediate in imine hydrolysis. The observed solvent isotope effect indicates that proton transfer also contributes to rate limitation. A concerted proton and hydride transfer is suggested by multiple substrate/solvent isotope effects, as well as a proton transfer in another step, likely hydrolysis of the carbinolamine. In agreement, dome-shaped proton inventories are observed for V2 and V2/K(Lys), suggesting that proton transfer exists in at least two sequential transition states.     

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.     

A product-inhibition study of bovine liver glutamate dehydrogenase.

1. Initial rates of oxidative deamination of L-glutamate with NAD+ as coenzyme, and of reductive aminiation of 2-oxoglutarate with NADH as coenzyme, catalysed by bovine liver glutamate dehydrogenase were measured in 0.111 M-sodium phosphate buffer, pH 7, at 25 degrees C, in the absence and presence of product inhibitors. All 12 possible combinations of variable substrate and product inhibitor were used. 2. Strict competition was observed between NAD+ and NADH, and between glutamate and 2-oxoglutarate. All other inhibition patterns were clearly non-competitive, except for inhibition by NH4+ with NAD+ as variable substrate. Here the extrapolation did not permit a clear distinction between competitive and non-competitive inhibition. 3. Mutually non-competitive behaviour between glutamate and NH4+ indicates that these substrates can be bound at the active site simultaneously. 4. Primary Lineweaver-Burk plots and derived secondary plots of slopes and intercepts against inhibitor concentration were linear, with one exception: with 2-oxoglutarate as variable substrate, the replot of primary intercepts against inhibitory NAD+ concentration was curved. 5. Separate Ki values were evaluated for the effect of each product inhibitor on the individual terms in the reciprocal initial-rate equations. With this information it is possible to calculate rates for any combination of substrate concentrations within the experimental range with any concentration of a single product inhibitor. 6. The inhibition patterns are consistent with neither a simple compulsory-order mechanism nor a rapid-equilibrium random-order mechanism without modification. They can, however, be reconciled with either type of mechanism by postulating appropirate abortive complexes. Of the two compulsory sequences that have been proposed, one, that in which the order of binding is NADH, NH4+, 2-oxoglutarate, requires an implausible pattern of abortive complex-formation to account for the results. 7. On the basis of a rapid-equilibrium random-order mechanism, dissociation constants can be calculated from the Ki values. Where these can be compared with independent estimates from the kinetics of the uninhibited reaction or from direct measurements of substrate binding, the agreement is reasonable good. On balance, therefore, the results provide further support for the rapid-equilibrium random-order mechanism under these conditions.     

STEADY STATE KINETICS OF RAT BRAIN PYRUVATE DEHYDROGENASE MULTIENZYME COMPLEX

Abstract— The overall steady state kinetic mechanism of pyruvate dehydrogenase multienzyme complex purified from rat brain has been investigated. Initial rate patterns were a series of parallel lines regardless of which substrate was varied at several fixed concentrations of other substrates. Product inhibition patterns showed that acetyl CoA is competitive vs CoA, that NADH is competitive vs NAD, and that both acetyl CoA and NADH are uncompetitive vs pyruvate. Both acetyl CoA and NADH are noncompetitive vs NAD and CoASH, respectively. These results are inconsistent with classical 'hexa uni' ping-pong mechanisms, but are consistent with a non-classical 3-site ping-pong mechanism.     

Enzymatic measurement of saccharopine with saccharopine dehydrogenase

We have developed an enzymatic method for measuring saccharopine, a key intermediate in lysine metabolism. With the enzyme saccharopine dehydrogenase, saccharopine can be oxidized to lysine and alpha-ketoglutarate with the corresponding conversion of NAD to NADH. The natural equilibrium favors saccharopine formation, but using hydrazine to trap one of the products, alpha-ketoglutarate, shifts the reaction toward quantitative oxidation of saccharopine. A stable endpoint is reached in 15-20 min, and although high concentrations of alpha-ketoglutarate slow the reaction, the end product is fully recovered. Unlike previous assays this technique is specific, convenient, and capable of measuring saccharopine directly in protein-free biological fluids or extracts.     

Determinants of substrate specificity for saccharopine dehydrogenase from Saccharomyces cerevisiae

A survey of NADH, alpha-Kg, and lysine analogues has been undertaken in an attempt to define the substrate specificity of saccharopine dehydrogenase and to identify functional groups on all substrates and dinucleotides important for substrate binding. A number of NAD analogues, including NADP, 3-acetylpyridine adenine dinucleotide (3-APAD), 3-pyridinealdehyde adenine dinucleotide (3-PAAD), and thionicotinamide adenine dinucleotide (thio-NAD), can serve as a substrate in the oxidative deamination reaction, as can a number of alpha-keto analogues, including glyoxylate, pyruvate, alpha-ketobutyrate, alpha-ketovalerate, alpha-ketomalonate, and alpha-ketoadipate. Inhibition studies using nucleotide analogues suggest that the majority of the binding energy of the dinucleotides comes from the AMP portion and that distinctly different conformations are generated upon binding of the oxidized and reduced dinucleotides. Addition of the 2'-phosphate as in NADPH causes poor binding of subsequent substrates but has little effect on coenzyme binding and catalysis. In addition, the 10-fold decrease in affinity of 3-APAD in comparison to NAD suggests that the nicotinamide ring binding pocket is hydrophilic. Extensive inhibition studies using aliphatic and aromatic keto acid analogues have been carried out to gain insight into the keto acid binding pocket. Data suggest that a side chain with three carbons (from the alpha-keto group up to and including the side chain carboxylate) is optimal. In addition, the distance between the C1-C2 unit and the C5 carboxylate of the alpha-keto acid is also important for binding; the alpha-oxo group contributes a factor of 10 to affinity. The keto acid binding pocket is relatively large and flexible and can accommodate the bulky aromatic ring of a pyridine dicarboxylic acid and a negative charge at the C3 but not the C4 position. However, the amino acid binding site is hydrophobic, and the optimal length of the hydrophobic portion of the amino acid carbon side chain is three or four carbons. In addition, the amino acid binding pocket can accommodate a branch at the gamma-carbon, but not at the beta-carbon.     

Intra- and intermolecular electron transfer processes in redox proteins

Initial velocity and product inhibition experiments were performed to characterize the kinetic mechanism of branched chain ketoacid dehydrogenase (the branched chain complex) activity. The results were directly compared to predicted patterns for a three-site ping-pong mechanism. Product inhibition experiments confirmed that NADH is competitive versus NAD+ and isovaleryl CoA is competitive versus CoA. Furthermore, both NADH and isovaleryl CoA were uncompetitive versus ketoisovaleric acid. These results are consistent with a ping-pong mechanism and are similar to pyruvate dehydrogenase and alpha-ketoglutarate dehydrogenase. However, inhibition patterns for isovaleryl CoA versus NAD+ and NADH versus CoA are not consistent with a ping-pong mechanism. These patterns may result from a steric interaction between the flavoprotein and transacetylase subunits of the complex. To determine the kinetic mechanism of the substrates and feedback inhibitors (NADH and isovaleryl CoA) of the branched chain complex, it was necessary to define the interaction of the inhibitors at nonsaturating fixed substrate (CoA and NAD+) concentrations. While the competitive inhibition patterns were maintained, slope replots for NADH versus NAD+ at nonsaturating CoA concentrations were parabolic. This unexpected finding resembles a linear mixed type of inhibition where the inhibition is a combination of pure competitive and noncompetitive inhibition.     

Influence of NAD-linked dehydrogenase activity on flux through oxidative phosphorylation.

1. We have examined systematically the relationship between the percentage reduction of cardiac mitochondrial NAD and the flux through oxidative phosphorylation, as measured by O2 uptake. Reduction of NAD was varied by varying the concentration of palmitoyl-L-carnitine, pyruvate, 2-oxoglutarate or glutamate in the presence of malate as the oxidizable substrate. 2. In the presence of ADP (State 3 respiration) there was a substantially linear positive relationship between O2 uptake and the percentage reduction of NAD. Coupled respiration in the absence of ADP also showed an increase with increasing NADH, with the exact shape of the relationship being variable. 3. When pyruvate and 2-oxoglutarate dehydrogenase activity were increased by increasing medium Ca2+ concentration within the range 5 nM to 1.23 microM, at non-saturating substrate concentrations, there was again a positive relationship between O2 uptake and the reduction of NAD; however, rates of O2 uptake tended to be higher at given values of NAD reduction when the incubation medium contained Ca2+. This is taken to indicate an activation by Ca2+ of the enzymes of phosphorylation or of the respiratory chain, in addition to the dehydrogenase activation. 4. When carboxyatractyloside plus ADP were used to generate 50% State 3 rates of O2 uptake with pyruvate or 2-oxoglutarate, sensitivity to Ca2+ was retained. However, when oligomycin plus 1 mM-ADP and 1 mM-ATP were used to generate 50% State 3, no such dependence was seen. 5. The results are interpreted to indicate a substantial role for substrate dehydrogenation in the overall regulation of oxidative phosphorylation when substrates are available at near-physiological concentrations.     

Microbial metabolism of amino alcohols. Formation of coenzyme B12-dependent ethanolamine ammonia-lyase and its concerted induction in Escherichia coli.

1. A branched-chain 2-oxo acid dehydrogenase was partially purified from ox liver mitochondria. 2. The preparation oxidized 4-methyl-2-oxopentanoate, 3-methyl-2-oxobutyrate and D- and L-3-methyl-2-oxopentanoate. The apparent Km values for the oxo acids and for thiamin pyrophosphate, CoA, NAD+ and Mg2+ were determined. 3. The oxidation of each oxo acid was inhibited by isovaleryl (3-methylbutyryl)-CoA (competitive with CoA) and by NADH (competitive with NAD+); Ki values were determined. 4. The preparation showed substrate inhibition with each 2-oxo acid. The oxidative decarboxylation of 4-methyl-2-oxo[1-14C]pentanoate was inhibited by 3-methyl-2-oxobutyrate and DL-3-methyl-2-oxopentanoate, but not by pyruvate. The Vmax. with 3-methyl-2-oxobutyrate as variable substrate was not increased by the presence of each of the other 2-oxo acids. 5. Ox heart pyruvate dehydrogenase did not oxidize these branched-chain 2-oxo acids and it was not inhibited by isovaleryl-CoA. The branched-chain 2-oxo acid dehydrogenase activity (unlike that of pyruvate dehydrogenase) was not inhibited by acetyl-CoA. 6. It is concluded that the branched-chain 2-oxo acid dehydrogenase activity is distinct from that of pyruvate dehydrogenase, and that a single complex may oxidize all three branched-chain 2-oxo acids.     

Metabolism and transport of glutamine and glucose in vascularly perfused small intestine of the rat

1. The proportion of active (dephosphorylated) pyruvate dehydrogenase in rat heart mitochondria was correlated with total concentration ratios of ATP/ADP, NADH/NAD+ and acetyl-CoA/CoA. These metabolites were measured with ATP-dependent and NADH-dependent luciferases. 2. Increase in the concentration ratio of NADH/NAD+ at constant [ATP]/[ADP] and [acetyl-CoA]/[CoA] was associated with increased phosphorylation and inactivation of pyruvate dehydrogenase. This was based on comparison between mitochondria incubated with 0.4mM- or 1mM-succinate and mitochondria incubated with 0.4mM-succinate+/-rotenone. 3. Increase in the concentration ratio acetyl-CoA/CoA at constant [ATP]/[ADP] and [NADH][NAD+] was associated with increased phosphorylation and inactivation of pyruvate dehydrogenase. This was based on comparison between incubations in 50 micrometer-palmitotoyl-L-carnitine and in 250 micrometer-2-oxoglutarate +50 micrometer-L-malate. 4. These findings are consistent with activation of the pyruvate dehydrogenase kinase reaction by high ratios of [NADH]/[NAD+] and of [acetyl-CoA]/[CoA]. 5. Comparison between mitochondria from hearts of diabetic and non-diabetic rats shows that phosphorylation and inactivation of pyruvate dehydrogenase is enhanced in alloxan-diabetes by some factor other than concentration ratios of ATP/ADP, NADH/NAD+ or acetyl-CoA/CoA.     

Kinetic mechanism of the hydroxypyruvate-lactate dehydrogenase-NADH system.

Chicken liver lactate dehydrogenase L-lactate : NAD+ oxidoreductase, EC1.1.1.27) reversibly catalyses the conversion of hydroxypyruvate to L-glycerate. The variation of the initial reaction rate with the substrate or coenzyme (NADH) concentration together with the inhibition caused by the reaction products and excess substrates, reveal that the kinetic mechanism of the reaction, with hydroxypyruvate as substrate, is of the rapid-equilibrium, ordered-ternary-complex type; NADH is the first substrate in the reaction sequence. Rate equations have been developed for the hydroxypyruvate.E.NADH system without inhibitors, with excess substrates, and with reaction products. Comparison of the rate equations obtained with those calculated theoretically from an ordered-ternary-complex mechanism reveals the existence of E.NAD.NADH,E.NAD-hydroxypyruvate and E.hydroxypyruvate complexes.     

Inactivation of 2-oxoglutarate dehydrogenase in rat liver mitochondria by its substrate and t-butyl hydroperoxide

Ketoacid oxidation in rat liver mitochondria was very sensitive to t-butyl hydroperoxide (t-BuOOH). Furthermore, 2-oxoglutarate and pyruvate each enhanced t-BuOOH-induced oxidative stresses of mitochondria, such as oxidation of pyridine nucleotides and GSH, inhibition of respiration with the other NAD-linked substrates, and peroxidation of mitochondrial lipids. We provide evidence that the t-BuOOH and ketoacid-induced effects are due to the failure of supply of NADH by 2-oxoglutarate dehydrogenase, and report the inactivation of the dehydrogenase in mitochondria by simultaneous addition of 2-oxoglutarate and t-BuOOH. Using the purified enzyme, we confirmed that t-BuOOH-induced inactivation of 2-oxoglutarate dehydrogenase was enhanced by its substrate and thiamine pyrophosphate protected the dehydrogenase from the inactivation. In contrast, succinate-dependent oxidation of mitochondria was not only scarcely affected by t-BuOOH, but also succinate protected against inactivation of 2-oxoglutarate dehydrogenase by t-BuOOH in mitochondria.     

Kinetic and stereochemical characterization of hamster liver 3 alpha-hydroxysteroid dehydrogenase and 3 alpha(17 beta)-hydroxysteroid dehydrogenase

The kinetic mechanism of NADP(+)-dependent 3 alpha-hydroxysteroid dehydrogenase and NAD(+)-dependent 3 alpha(17 beta)-hydroxysteroid dehydrogenase, purified from hamster liver cytosol, was studied in both directions. For 3 alpha-hydroxysteroid dehydrogenase, the initial velocity and product inhibition studies indicated that the enzyme reaction sequence is ordered with NADP+ binding to the free enzyme and NADPH being the last product to be released. Inhibition patterns by Cibacron blue and hexestrol, and binding studies of coenzyme and substrate are also consistent with an ordered bi bi mechanism. For 3 alpha(17 beta)-hydroxysteroid dehydrogenase, the steady-state kinetic measurements and substrate binding studies suggest a random binding pattern of the substrates and an ordered release of product; NADH is released last. However, the two enzymes transferred the pro-R-hydrogen atom of NAD(P)H to the carbonyl substrate.     

Kinetic characterization of the pyruvate and oxoglutarate dehydrogenase complexes from human heart

The Michaelis constant values for the highly purified pyruvate dehydrogenase complex (PDC) from human heart are 25, 13 and 50 microM for pyruvate, CoA and NAD, respectively. Acetyl-CoA produces a competitive inhibition of PDC (Ki = 35 microM) with respect to CoA, whereas NADH produces the same type of inhibition with respect to NAD (Ki = 36 microM). The oxoglutarate dehydrogenase complex (OGDC) from human heart has active sites with two different affinities for 2-oxoglutarate ([S]0.5 of 30 and 120 microM). ADP (1 mM) decreases the [S]0.5 values by a half. The inhibition of OGDC (Ki = 81 microM) by succinyl-CoA is of a competitive type with respect to CoA (Km = 2.5 microM), whereas that of NADH (Ki = 25 microM) is of a mixed type with respect to NAD (Km = 170 microM).     

Inhibition of lipogenesis by halothane in isolated rat liver cells.

1. Halothane at clinically effective concentrations [2.5 and 4% (v/v) of the gas phase of the incubation flask] was found to inhibit significantly lipogenesis from endogenous substrates, e.g., glycogen, or from added lactate plus pyruvate. This was accompanied by a decrease in the ratio of the free [NAD+]/[NADH] of the mitochondrion and the cytoplasm, as shown by the [3-hydroxybutyrate]/[acetoacetate] ratio and the [lactate]/[pyruvate] ratio. 2. Acetoacetate or pyruvate decreased the inhibitory effect of halothane and restored lipogenesis to control rates. They were reduced rapidly by 3-hydroxybutyrate dehydrogenase or lactate dehydrogenase respectively, with the concomitant oxidation of NADH and the generation of NAD+. 3. These results suggest that the mechanism by which halothane inhibits lipogenesis from glycogen or lactate is by inhibition of the oxidation of NADH; this results in inhibition of flux of carbon through pyruvate dehydrogenase and a shortage of acetyl-CoA for fatty acid synthesis. Thus when NADH acceptors are added in the presence of halothane, the concentration of mitochondrial NAD+ is raised so that the flux of carbon through pyruvate dehydrogenase increases and lipogenesis is restored.