Combined coenzyme-substrate analogues of various dehydrogenases. Synthesis of (3S)- and (3R)-5-(3-carboxy-3-hydroxypropyl)nicotinamide adenine dinucleotide and their interaction with (S)- and (R)-lactate-specific dehydrogenases.

Two diastereomeric nicotinamide adenine dinucleotide (NAD+) derivatives were synthesized in which the substrates of (S)-and (R)-lactate-specific dehydrogenases are covalently attached via a methylene spacer at position 5 of the nicotinamide ring. The corresponding nicotinamide derivatives were obtained stereospecifically by enzymatic reduction of 5-(2-oxalylethyl)nicotinamide. (3S)-5-(3-Carboxy-3-hydroxypropyl)-NAD+ undergoes and intramolecular hydride transfer in the presence of pig heart lactate dehydrogenase, forming the corresponding coenzyme-substrate analogue composed of pyruvate and NADH. No cross-reaction products resulting from an intermolecular reaction are observed. Two (R)-lactate specific dehydrogenases, however, do not catalyze a similar reaction in either one of the two diastereomers. A possible arrangement of the substrates in the active centers of these enzymes is proposed. 5-Methyl-NAD+ and 5-methyl-NADH are active coenzymes of pig heart lactate dehydrogenase in contrast to reports in the literature. (S)-Lactate binds to this enzyme in the absence of coenzyme, exhibiting a dissociation constant of 11 mM.     

Intramolecular hydride transfer of a combined coenzyme-substrate analog by D- and L-lactate dehydrogenases

The synthesis of 3-[(3-carboxy-3-oxopropyl)aminocarbonyl]pyridine adenine dinucleotide, a new combined analog of NADH and pyruvate with pyruvate covalently attached to the amide nitrogen atom of the dihydronicotinamide ring via an additional methylene group, is described. In the presence of D-lactate dehydrogenase from Limulus polyphemus, from Lactobacillus leichmannii, and L-lactate dehydrogenase from pig skeletal muscle a redox reaction takes place between the pyruvate moiety and the dihydropyridine ring of the analog. This reaction is shown to be intramolecular by competition experiments with pyruvate. Degradation of the reaction products reveals that the carbon-2 atom of the formed lactate side chain exhibits D configuration in each of these cases studied.     

The reversibility of cytosolic dehydrogenase reactions in hepatocytes from starved and fed rats. Effect of fructose.

The metabolism of [2-3H]lactate was studied in isolated hepatocytes from fed and starved rats metabolizing ethanol and lactate in the absence and presence of fructose. The yields of 3H in ethanol, water, glucose and glycerol were determined. The rate of ethanol oxidation (3 mumol/min per g wet wt.) was the same for fed and starved rats with and without fructose. From the detritiation of labelled lactate and the labelling pattern of ethanol and glucose, we calculated the rate of reoxidation of NADH catalysed by lactate dehydrogenase, alcohol dehydrogenase and triosephosphate dehydrogenase. The calculated flux of reducing equivalents from NADH to pyruvate was of the same order of magnitude as previously found with [3H]ethanol or [3H]xylitol as the labelled substrate [Vind & Grunnet (1982) Biochim. Biophys. Acta 720, 295-302]. The results suggest that the cytoplasm can be regarded as a single compartment with respect to NAD(H). The rate of reduction of acetaldehyde and pyruvate was correlated with the concentration of these metabolites and NADH, and was highest in fed rats and during fructose metabolism. The rate of reoxidation of NADH catalysed by lactate dehydrogenase was only a few per cent of the maximal activity of the enzymes, but the rate of reoxidation of NADH catalysed by alcohol dehydrogenase was equal to or higher than the maximal activity as measured in vitro, suggesting that the dissociation of enzyme-bound NAD+ as well as NADH may be rate-limiting steps in the alcohol dehydrogenase reaction.     

Chirality of the hydrogen transfer to the coenzyme catalyzed by ribitol dehydrogenase from Klebsiella pneumoniae and D-mannitol 1-phosphate dehydrogenase from Escherichia coli.

The stereochemistry of the hydrogen transfer to NAD catalyzed by ribitol dehydrogenase (ribitol:NAD 2-oxidoreductase, EC 1.1.1.56) from Klebsiella pneumoniae and D-mannitol-1-phosphate dehydrogenase (D-mannitol-1-phosphate:NAD 2-oxidoreductase, EC 1.1.1.17) from Escherichia coli was investigated. [4-3H]NAD was enzymatically reduced with nonlabelled ribitol in the presence of ribitol dehydrogenase and with nonlabelled D-mannitol 1-phosphate and D-mannitol 1-phosphate dehydrogenase, respectively. In both cases the [4-3H]-NADH produced was isolated and the chirality at the C-4 position determined. It was found that after the transfer of hydride, the label was in both reactions exclusively confined to the (4R) position of the newly formed [4-3H]NADH. In order to explain these results, the hydrogen transferred from the nonlabelled substrates to [4-3H]NAD must have entered the (4S) position of the nicotinamide ring. These data indicate for both investigated inducible dehydrogenases a classification as B or (S) type enzymes. Ribitol also can be dehydrogenated by the constitutive A-type L-iditol dehydrogenase (L-iditol:NAD 5-oxidoreductase, EC 1.1.1.14) from sheep liver. When L-iditol dehydrogenase utilizes ribitol as hydrogen donor, the same A-type classification for this oxidoreductase, as expected, holds true. For the first time, opposite chirality of hydrogen transfer to NAD in one organic reaction--ribitol + NAD = D-ribu + NADH + H--is observed when two different dehydrogenases, the inducible ribitol dehydrogenase from K. pneumoniae and the constitutive L-iditol dehydrogenase from sheep liver, are used as enzymes. This result contradicts the previous generalization that the chirality of hydrogen transfer to the coenzyme for the same reaction is independent of the source of the catalyzing enzyme.     

Effect of ATP on purified L(+) lactate dehydrogenase from electric organ of Electrophorus electricus (L.)

L(+) lactate dehydrogenase (LDH) activity from the electric organ of Electrophorus electricus was measured in the presence of ATP in the forward (substrate lactate) and reverse (substrate pyruvate) enzymatic reactions. The I50 for ATP was first determined and then the kinetics of the reactions were investigated with either constant coenzyme (NAD or NADH) concentration and varying substrate (lactate or pyruvate) concentration, or, constant substrate and varying coenzyme concentration. The kinetic data showed that ATP inhibits LDH uncompetitively with respect to the reduced and the oxidized coenzyme. As for the substrates, ATP gives a mixed type inhibition for lactate and a noncompetitive inhibition for pyruvate.     

Involvement of pyruvate dehydrogenase in product formation in pyruvate-limited anaerobic chemostat cultures of Enterococcus faecalis NCTC 775

Enterococcus faecalis NCTC 775 was grown anaerobically in chemostat culture with pyruvate as the energy source. At low culture pH values, high in vivo and in vitro activities were found for both pyruvate dehydrogenase and lactate dehydrogenase. At high culture pH values the carbon flux was shifted towards pyruvate formate lyase. Some mechanisms possibly involved in this metabolic switch are discussed. In particular attention is paid to the NADH/NAD ratio (redox potential) and the fructose-1,6-bisphosphate-dependent lactate dehydrogenase activity as possible regulatory factors.Abbreviations PDH pyruvate dehydrogenase complex (EC 1.2.2.2) - PFL pyruvate formate lyase (EC 2.3.1.54) - LDH lactate dehydrogenase (EC 1.1.1.27) - FBP fructose-1,6-bisphosphate - MTT 3-(4,5-dimethyl-thiazoyl-2)-2,5-diphenyltetrazolium bromide - TPP thiamine pyrophosphate     

Substrate-induced conformational changes in lactate dehydrogenase. Proteolysis of the immobilized enzyme in the presence of specific substrates.

We report here a new approach to the study of the conformation of enzymes in the presence of specific substrates. Rabbit muscle lactate dehydrogenase was attached to CL-Sepharose via a cleavable spacer arm (-NH-(CH2)6NHCO(CH2)2SS(CH2)2CO-). The bound lactate dehydrogenase was digested with subtilisin BPN' in the presence of substrates of lactate dehydrogenase. The use of a flow system permits the maintenance of saturating levels of substrates. Proteolysis was followed by loss of activity of the enzyme column. The time course of proteolysis in the presence of either NADH, NAD+, or pyruvate alone did not differ from the control. However, when NADH and pyruvate were present simultaneously, the enzyme became more susceptible to proteolysis. The initial rate of proteolysis was increased by 40%. The abortive ternary complex (lactate dehydrogenase - NAD+ - pyruvate) also showed an increase in susceptibility to proteolysis. These findings clearly show that the productive ternary complex (lactate dehydrogenase - NADH - pyruvate) is conformationally different from the apoenzyme and binary complexes under optimal catalytic conditions.     

Thermodynamic studies of the activation of rabbit muscle lactate dehydrogenase by phosphate.

In an attempt to trace the source of phosphate activation of the enzyme-catalysed pyruvate-lactate interconversion by rabbit muscle lactate dehydrogenase, equilibrium constants were measured to examine the effects of phosphate on interactions pertinent to the enzymic process. Frontal gel-chromatographic studies of the binding of NADH to the enzyme established that the intrinsic association constant is doubled in the presence of 50 mM-phosphate in the buffer (pH 7.4, I0.15). From kinetic studies of the competition between NAD+ and NADH for the coenzyme-binding sites of the enzyme it is concluded that the binding of oxidized nicotinamide nucleotide is also doubled in the presence of 50 mM-phosphate. Competitive-inhibition studies and fluorescence-quenching measurements indicated the lack of a phosphate effect on ternary-complex formation between enzyme-NADH complex and oxamate, a substrate analogue of pyruvate. The equilibrium constant for the interaction between enzyme-NAD+ complex and oxalate, an analogue of lactate, was also shown, by difference spectroscopy, to be insensitive to phosphate concentration. Provided that the effects observed with the substrate analogues mimic those operative in the kinetic situation, the equilibrium constant governing the isomerization of ternary complex is also independent of phosphate concentration. It is concluded that enhanced coenzyme binding is the source of phosphate activation of the rabbit muscle lactate dehydrogenase system.     

Pig heart lactate dehydrogenase. Binding of pyruvate and the interconversion of pyruvate-containing ternary complexes.

1. Lactate oxidation catalysed by pig heart lactate dehydrogenase was studied in the presence of inhibitory concentrations of pyruvate. Experimental results show the presence of an intermediate which occurs immediately after the hydride transfer step, but before the dissociation of pyruvate and the H+ produced by the reaction. The rate constant for pyruvate dissociation and the dissociation constant for pyruvate from the ternary complex differ from those obtained in pyruvate reduction experiments. 2.In single-turnover pyruvate reduction by pig heart lactate dehydrogenase at pH8.0 pyruvate can bind to the enzyme before a H+ is taken up, and the subsequent uptake of a H+ is governed by a step that is also rate-limiting for single-turnover and steady-state NADH oxidation. 3. Observation of various intermediates in the single-turnover pyruvate reduction experiments has made it possible to determine separately the dissociation constant and Km value for pyruvate at pH8.0, and also the catalytic turnover rate and Km for pyruvate under first-order conditions at different pH values. 4. Further studies on single-turnover pyruvate reduction carried out in 2H2O, or in water at low temperature, show another step which, under these conditions, is slower than that controlling H+ uptake and rate-limiting for NADH oxidation. A scheme is presented which explains these results.     

Inhibition of Lactate Production in Rat Brain Extracts and Synaptosomes by 3-[4-(Reduced 3-Pyridine Aldehyde-Adenine Dinucleotide)]-Pyruvate

In basic solutions, pyruvate enolizes and reacts (through its 3-carbon) with the 4-carbon of the nicotinamide ring of NAD+, yielding an NAD-pyruvate adduct in which the nicotinamide ring is in the reduced form. This adduct is a strong inhibitor of lactate dehydrogenase, presumably because it binds simultaneously to the NADH and pyruvate sites. The potency of the inhibition, however, is muted by the adduct's tendency to cyclize to a lactam. We prepared solutions of the pyruvate adduct of NAD+ and of NAD+ analogues in which the -C(O)NH2 of NAD+ was replaced with -C(S)NH2, -C(O)CH3, and -C(O)H. Of the four, only the last analogue, 3-[4-(reduced 3-pyridine aldehyde-adenine dinucleotide)]-pyruvate (RAP) cannot cyclize and it was found to be the most potent inhibitor of beef heart and rat brain lactate dehydrogenases. The inhibitor binds very tightly to the NADH site (Ki approximately 1 nM for the A form). Even at high concentrations (20 microM), RAP had little or no effect on rat brain glyceraldehyde-3-phosphate, pyruvate, alpha-ketoglutarate, isocitrate, soluble and mitochondrial malate, and glutamate dehydrogenases. The glycolytic enzymes, hexokinase and phosphofructokinase, were similarly unaffected. RAP strongly inhibited lactate production from glucose in rat brain extracts but was less effective in inhibiting lactate production from glucose in synaptosomes.     

On the origin of the lactate dehydrogenase induced rate effect

To evaluate the ability of lactate dehydrogenase to facilitate the bond making/breaking steps for both the addition of pyruvate enol to NAD (pyruvate adduct reaction) and the normal redox reaction, the ability of the enzyme to facilitate the tautomerization of bound pyruvate is assessed. In addition, the equilibrium constants for the adduct reaction are obtained for both bound and free reactants from the ratio of the rate constants in the forward and reverse reactions (at pH 7). The latter comparison indicates that the enzyme facilitates bond making/breaking in the (forward) pyruvate adduct reaction by a factor of about 10(11) M. Similar comparisons suggest that reactant immobilization accounts for about 1000 M of this 10(11) M rate effect. Since the (pH-independent) rate constant for the ketonization of bound pyruvate enol assisted by the external buffer, imidazolium ion, is 2 X 10(7) M-1 s-1 and the corresponding rate constant for free pyruvate enol, again assisted by imidazolium ion, is 35 M-1 s-1 [Burger, J. W., II, & Ray, W. J., Jr. (1978) Biochemistry 17, 1664], the enzyme facilitates the bond making/breaking steps associated with the conversion of bound HO-C less than to bound O = C less than by a factor of about 10(6)-fold. The product of the above two rate enhancement factors and the rate factor suggested previously for the environmental effect on NAD produced by its binding to lactate dehydrogenase, 100-fold, is 10(11) M, and it accounts for the bond making/breaking effects exerted by the enzyme in the pyruvate adduct reaction. The rate constant for oxidation of ethanol (a model for lactate) by 1-methylnicotinamide (a model for NAD) is about 5 X 10(-12) M-1 s-1 at 25 degrees C in pure ethanol (delta H for this reaction is about 30 kcal/mol). The ratio of the rate constants for E X NAD X Lac----E X NADH X Pyr and the above model reaction is estimated as about 10(14) M in water; i.e., the LDH-induced rate effect is about 10(14) M. The product of the values for the above rate factors for the normal redox reaction is about 10(12) M. Although the value of this product is less certain than that for the adduct reaction, these rate factors do account for much of the LDH-induced rate effect.     

The redox switch/redox coupling hypothesis

We provide an integrative interpretation of neuroglial metabolic coupling including the presence of subcellular compartmentation of pyruvate and monocarboxylate recycling through the plasma membrane of both neurons and glial cells. The subcellular compartmentation of pyruvate allows neurons and astrocytes to select between glucose and lactate as alternative substrates, depending on their relative extracellular concentration and the operation of a redox switch. This mechanism is based on the inhibition of glycolysis at the level of glyceraldehyde 3-phosphate dehydrogenase by NAD(+) limitation, under sufficiently reduced cytosolic NAD(+)/NADH redox conditions. Lactate and pyruvate recycling through the plasma membrane allows the return to the extracellular medium of cytosolic monocarboxylates enabling their transcellular, reversible, exchange between neurons and astrocytes. Together, intracellular pyruvate compartmentation and monocarboxylate recycling result in an effective transcellular coupling between the cytosolic NAD(+)/NADH redox states of both neurons and glial cells. Following glutamatergic neurotransmission, increased glutamate uptake by the astrocytes is proposed to augment glycolysis and tricarboxylic acid cycle activity, balancing to a reduced cytosolic NAD(+)/NADH in the glia. Reducing equivalents are transferred then to the neuron resulting in a reduced neuronal NAD(+)/NADH redox state. This may eventually switch off neuronal glycolysis, favoring the oxidation of extracellular lactate in the lactate dehydrogenase (LDH) equilibrium and in the neuronal tricarboxylic acid cycles. Finally, pyruvate derived from neuronal lactate oxidation, may return to the extracellular space and to the astrocyte, restoring the basal redox state and beginning a new loop of the lactate/pyruvate transcellular coupling cycle. Transcellular redox coupling operates through the plasma membrane transporters of monocarboxylates, similarly to the intracellular redox shuttles coupling the cytosolic and mitochondrial redox states through the transporters of the inner mitochondrial membrane. Finally, transcellular redox coupling mechanisms may couple glycolytic and oxidative zones in other heterogeneous tissues including muscle and tumors.     

NAD(H) enhances the Cu(II)-mediated inactivation of lactate dehydrogenase by increasing the accessibility of sulfhydryl groups

Copper ions are known to inactivate a variety of enzymes, and lactate dehydrogenase (LDH) is exceptionally sensitive to the presence of this metal. We now found that NADH strongly enhances the Cu(II)-mediated loss of LDH activity. Surprisingly, NADH was not oxidized in this process and also NAD⁺ promoted the Cu(II)-dependent inactivation of LDH. Catalase only partly protected the enzyme, whereas hypoxia even enhanced LDH inactivation. NAD(H) accelerated sulfhydryl (SH) group oxidation of LDH by 5,5-dithio-bis(2-nitrobenzoic acid) (DTNB), and, vice versa, LDH-mediated Cu(II) reduction. LDH activity was preserved by thiol donators and pyruvate and partially preserved by lactate and oxamate. Our results suggest that reactive oxygen species (ROS) are of minor importance for the inactivation of LDH induced by Cu(II)/NADH. We propose that conformational changes of the enzymes' active sites induced by NAD(H)-binding increase the accessibility of active sites' cysteine residues to Cu(II) thereby accelerating their oxidation and, consequently, loss of catalytic activity.     

The rates of defined changes in protein structure during the catalytic cycle of lactate dehydrogenase

Rapid mixing, kinetic experiments were performed on native and modified [Tyr(3NO2)237)] porcine H4 lactate dehydrogenase at low temperatures in a medium containing 30% dimethyl sulphoxide. In the temperature range -16 to +8 degrees C, the modified enzyme-NADH complex, when mixed with 1 mM pyruvate, is converted to enzyme, NAD+ and lactate at two distinctly different rates. At -16 degrees C the more rapid process occurs at a rate of 40 s-1 and the slower at 3 s-1. The slower rate is identical to that assigned to the steady-state turnover of the enzyme in these conditions and therefore reflects the slow, rate-limiting rearrangement of protein structure which has been inferred from previous kinetic experiments. The fast phase of NADH oxidation, however, proceeds at a rate which coincides with that of the closure of a loop of polypeptide over the active site of the enzyme (sensed by the nitrotyrosine group, which protonates in response to the approach of glutamate 107, a residue situated on this mobile loop). We explain these results by proposing that: (i) both the slow and fast changes in protein structure must occur before the enzyme can accomplish the redox step, (ii) the enzyme-NADH (binary) complex exists in two, slowly interconverting forms, (iii) the structural change giving rise to this slow conformational equilibrium can also occur in the ternary (enzyme-NADH-pyruvate) complex and (iv) it is this step which limits the rate of the steady-state reaction. Both of the binary forms are able to bind pyruvate, but the rate of NADH oxidation in one of the forms is rapid, since it has already undergone this slow rearrangement. In this rapidly reacting form, it is the closure of the loop (not transfer of the hydride ion) which limits the rate at which the coenzyme is oxidized, while the slowly reacting form must undergo both loop-closure and the slow structural conversion before the redox reaction can occur.     

The lipoamide dehydrogenase from Mycobacterium tuberculosis permits the direct observation of flavin intermediates in catalysis

Lipoamide dehydrogenase catalyses the NAD(+)-dependent oxidation of the dihydrolipoyl cofactors that are covalently attached to the acyltransferase components of the pyruvate dehydrogenase, alpha-ketoglutarate dehydrogenase, and glycine reductase multienzyme complexes. It contains a tightly, but noncovalently, bound FAD and a redox-active disulfide, which cycle between the oxidized and reduced forms during catalysis. The mechanism of reduction of the Mycobacterium tuberculosis lipoamide dehydrogenase by NADH and [4S-(2)H]-NADH was studied anaerobically at 4 degrees C and pH 7.5 by stopped-flow spectrophotometry. Three phases of enzyme reduction were observed. The first phase, characterized by a decrease in absorbance at 400-500 nm and an increase in absorbance at 550-700 nm, was fast (k(for) = 1260 s(-)(1), k(rev) = 590 s(-)(1)) and represents the formation of FADH(2).NAD(+), an intermediate that has never been observed before in any wild-type lipoamide dehydrogenase. A primary deuterium kinetic isotope effect [(D)(k(for) + k(rev)) approximately 4.2] was observed on this phase. The second phase, characterized by regain of the absorbance at 400-500 nm, loss of the 550-700 nm absorbance, and gain of 500-550 nm absorbance, was slower (k(obs) = 200 s(-)(1)). This phase represents the intramolecular transfer of electrons from FADH(2) to the redox-active disulfide to generate the anaerobically stable two-electron reduced enzyme, EH(2). The third phase, characterized by a decrease in absorbance at 400-550 nm, represents the formation of the four-electron reduced form of the enzyme, EH(4). The observed rate constant for this phase showed a decreasing NADH concentration dependence, and results from the slow (k(for) = 57 s(-)(1), k(rev) = 128 s(-)(1)) isomerization of EH(2) or slow release of NAD(+) before rapid NADH binding and reaction to form EH(4). The mechanism of oxidation of EH(2) by NAD(+) was also investigated under the same conditions. The 530 nm charge-transfer absorbance of EH(2) shifted to 600 nm upon NAD(+) binding in the dead time of mixing of the stopped-flow instrument and represents formation of the EH(2).NAD(+) complex. This was followed by two phases. The first phase (k(obs) = 750 s(-)(1)), characterized by a small decrease in absorbance at 435 and 458 nm, probably represents limited accumulation of FADH(2).NAD(+). The second phase was characterized by an increase in absorbance at 435 and 458 nm and a decrease in absorbance at 530 and 670 nm. The observed rate constant that describes this phase of approximately 115 s(-)(1) probably represents the overall rate of formation of E(ox) and NADH from EH(2) and NAD(+), and is largely determined by the slower rates of the coupled sequence of reactions preceding flavin oxidation.     

Transient-kinetic studies of pig muscle lactate dehydrogenase

1. The very fast pre-steady-state formation of NADH catalysed by pig M(4) lactate dehydrogenase was equivalent to the enzyme-site concentration at pH values greater than 8.0 and to one-half the site concentration at pH6.8. 2. The rate of dissociation of NADH from the enzyme at pH8.0 (450s(-1)) in the absence of other substrates is faster than the steady-state oxidation of lactate (80s(-1)). The latter process is therefore controlled by a step before NADH dissociation but subsequent to the hydride transfer. 3. The oxidation of enzyme-NADH by excess of pyruvate was studied as a first-order process at pH9.0. There was no effect of NADD on this reaction and it was concluded that the ternary complex undergoes a rate-limiting change before the hydride-transfer step. 4. Some conclusions about the reactions catalysed by the M(4) isoenzyme were drawn from a comparison of these results with those obtained with the H(4) isoenzyme and liver alcohol dehydrogenase.     

Selective inactivation of the transacylase components of the 2-oxo acid dehydrogenase multienzyme complexes of Escherichia coli.

1. The reaction of the pyruvate dehydrogenase multienzyme complex of Escherichia coli with maleimides was examined. In the absence of substrates, the complex showed little or no reaction with N-ethylmaleimide. However, in the presence of pyruvate and N-ethylmaleimide, inhibition of the pyruvate dehydrogenase complex was rapid. Modification of the enzyme was restricted to the transacetylase component and the inactivation was proportional to the extent of modification. The lipoamide dehydrogenase activity of the complex was unaffected by the treatment. The simplest explanation is that the lipoyl groups on the transacetylase are reductively acetylated by following the initial stages of the normal catalytic cycle, but are thereby made susceptible to modification. Attempts to characterize the reaction product strongly support this conclusion. 2. Similarly, in the presence of N-ethylmaleimide and NADH, much of the pyruvate dehydrogenase activity was lost within seconds, whereas the lipoamide dehydrogenase activity of the complex disappeared more slowly: the initial site of the reaction with the complex was found to be in the lipoyl transacetylase component. The simplest interpretation of these experiments is that NADH reduces the covalently bound lipoyl groups on the transacetylase by means of the associated lipoamide dehydrogenase component, thereby rendering them susceptible to modification. However, the dependence of the rate and extent of inactivation on NADH concentration was complex and it proved impossible to inhibit the pyruvate dehydrogenase activity completely without unacceptable modification of the other component enzymes. 3. The catalytic reduction of 5,5'-dithiobis-(2-nitrobenzoic acid) by NADH in the presence of the pyruvate dehydrogenase complex was demonstrated. A new mechanism for this reaction is proposed in which NADH causes reduction of the enzyme-bound lipoic acid by means of the associated lipoamide dehydrogenase component and the dihydrolipoamide is then oxidized back to the disulphide form by reaction with 5,5'-dithiobis-(2-nitrobenzoic acid). 4. A maleimide with a relatively bulky N-substituent, N-(4-diemthylamino-3,5-dinitrophenyl)maleimide, was an effective replacement for N-ethylmaleimide in these reactions with the pyruvate dehydrogenase complex. 5. The 2-oxoglutarate dehydrogenase complex of E. coli behaved very similarly to the pyruvate dehydrogenase complex, in accord with the generally accepted mechanisms of the two enzymes. 6. The treatment of the 2-oxo acid dehydrogenase complexes with maleimides in the presence of the appropriate 2-oxo acid substrate provides a simple method for selectively inhibiting the transacylase components and for introducing reporter groups on to the lipoyl groups covalently bound to those components.     

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.     

The inhibition of lactate dehydrogenase by hydrated pyruvate

The lactate dehydrogenase-catalyzed reduction of pyruvate by NADH was studied using a spectroscopic method. The inhibitory effect exhibited by high concentrations of pyruvate was investigated in phosphate and 2,2-diethylmalonate buffers. Kinetic studies were carried out in which the rate of the enzyme-catalyzed reaction was monitored at various stages of pyruvate hydration, H₂O + CH₃COCO₂^? ? CH₃C(OH)₂2C0₂^?. Buffered solutions of different initial relative amounts of ketopyruvate and hydrated pyruvate (2,2-dihydroxypropanoic acid) were also preincubated with the enzyme and NAD⁺. Kinetic runs were initiated in the resultant solutions at various stages of incubation by the introduction of NADH. The results of the present investigation indicate that hydrated pyruvate is a major inhibitor of lactate dehydrogenase and forms an inhibitory complex with the enzyme and oxidized coenzyme.     

Lactate dehydrogenase from gastrocnemius muscle of turtle

Five bands of lactate dehydrogenase (LDH) isoenzymes were seen by polyacrylamide gel electrophoresis in gastrocnemius muscle of the turtle (Kachuga smithi). The major band was of M2H2 type and was partially purified by gel filtration and affinity chromatography. The specific activity of the enzyme was 2.6 units/mg protein. The half-life of the enzyme at 4 degrees C, was about 7 days. The optimum temperature for enzyme activity was 30 degrees C and the enzyme was irreversibly inactivated at 40 degrees C. The optimum pH for the forward reaction (pyruvate to lactate) was 5.5, while for reverse reaction it was between 8.0 to 9.5. The apparent Km values for pyruvate, NADH, lactate and NAD+ were 0.20, 0.013, 25 and 0.333 mM, respectively. Oxalate was found to be the inhibitor of LDH with Ki of about 4.2 mM.