Kinetic study of the mechanisms of eliminating substrate inhibition of lactate dehydrogenase by anions and pH

The dependence of lactate dehydrogenase inhibition at high pyruvate concentrations on pH and neutral salt anions was studied. It was shown that Cl- anions compete with the substrate within the ternary inhibitory complex, ENADpyr in equilibrium ENADCl-, as a result of which the pyruvate-induced inhibition is eliminated. The KD values for Cl- (50 mM) and I- (27 mM) were calculated from the substrate velocity curves at high concentrations of pyruvate. It was supposed that pyruvate inhibition elimination by OH- proceeds via the same kinetic mechanism. The pK value (7.1 +/- 0.1) calculated from this model corresponds to pKn of essential His-195. The additivity of OH- and Cl- function was demonstrated.     

Regulation of pea mitochondrial pyruvate dehydrogenase complex activity: inhibition of ATP-dependent inactivation

In contrast to the pyruvate dehydrogenase complex (PDC) from animal mitochondria, our in situ and in vitro studies indicate that the ATP:ADP ratio has little or no effect in regulating the mitochondrial pyruvate dehydrogenase complex from green pea seedlings. Pyruvate was a competitive inhibitor of ATP-dependent inactivation (Ki = 59 microM), while the PDC had a Km for pyruvate of microM. Thiamine pyrophosphate, the coenzyme for the pyruvate dehydrogenase (PDH) component of the complex, did not inhibit ATP-dependent inactivation when used alone but it enhanced inhibition by pyruvate. As such, thiamine pyrophosphate was a competitive inhibitor (Ki = 130 nM) of ATP-dependent inactivation. A model is proposed for the pyruvate plus thiamine pyrophosphate inhibition of ATP-dependent inactivation of the pyruvate dehydrogenase complex in which pyruvate exerts its inhibition of inactivation by altering or protecting the protein substrate from phosphorylation and not by directly inhibiting PDH kinase.     

Octopine dehydrogenase from Pecten maximus: steady-state mechanism

The steady-state kinetic mechanism of the reaction catalyzed by octopine dehydrogenase [N2-(1-carboxyethyl)-L-arginine:NAD+ oxidoreductase] was investigated at pH 6.9 and 9.2 by studies of substrate inhibition, analogue inhibition, and product inhibition. In the direction of octopine synthesis, the inhibition patterns in the presence of delta- guanidinovalerate and propionate show that NADH binds to the enzyme first followed by L-arginine and pyruvate which bind randomly. In the direction of octopine oxidation, the substrate patterns show that NAD binds to the enzyme before octopine in a rapid equilibrium fashion, and the product inhibition patterns show that the products L-arginine and pyruvate are released in a random fashion. Double, synergistic, substrate inhibition by L-arginine and pyruvate was shown to be due to binding (hypothetically of the imine) to the free enzyme and the enzyme-NAD complex. Furthermore, an alternate minor pathway was demonstrated which includes an enzyme-NADH-octopine complex and an enzyme-octopine complex.     

Inhibition of human erythrocyte lactate dehydrogenase by high concentrations of pyruvate. Evidence for the competitive substrate inhibition.

The mechanism of the inhibitory effect of high concentrations of pyruvate on human erythrocyte lactate dehydrogenase has been studied by the use of a new parameter, delta, defined as the difference between the reciprocals of initial reaction rates obtained from experimental measurements and hypothetical linear Lineweaver-Burk plots. This parameter served as a method for differentiating between the competitive and umcompetitive substrate inhibition. Results of this study indicate that pyruvate is a competitive substrate inhibitor. It is suggested that the inhibitory effect of pyruvate is due to its competition with NADH for binding to the free enzyme and formation of an inactive enzyme-pyruvate binary complex. The competitive nature of pyruvate inhibition is further supported by the results of a kinetic study with NADH as the variable substrate. The dissociation constnat of the inactive enzyme-pyruvate binary complex was determined to be 101 micrometer. The physiological significance of the inhibitory effect could be to preserve a level of NADH concentration necessary for other vital enzymic reactions of living cells despite the presence of a high concentration of pyruvate.     

Kinetic mechanism of the endogenous lactate dehydrogenase activity of duck epsilon-crystallin

Initial velocity, product inhibition, and substrate inhibition studies suggest that the endogenous lactate dehydrogenase activity of duck epsilon-crystallin follows an order Bi-Bi sequential mechanism. In the forward reaction (pyruvate reduction), substrate inhibition by pyruvate was uncompetitive with inhibition constant of 6.7 +/- 1.7 mM. In the reverse reaction (lactate oxidation), substrate inhibition by L-lactate was uncompetitive with inhibition constant of 158 +/- 25 mM. The cause of these inhibitions may be due to epsilon-crystallin-NAD(+)-pyruvate and epsilon-crystallin-NADH-L-lactate abortive ternary complex formation as suggested by the multiple inhibition studies. Pyruvate binds to free enzyme very poorly, with a very large dissociation constant. Bromopyruvate, fluoropyruvate, pyruvate methyl ester, and pyruvate ethyl ester are alternative substrates for pyruvate. 3-Acetylpyridine adenine dinucleotide, nicotinamide 1,N6-ethenoadenine dinucleotide, and nicotinamide hypoxanthine dinucleotide serve as alternative coenzymes for epsilon-crystallin. All the above alternative substrates or coenzymes showed an intersecting initial-velocity pattern conforming to the order Bi--Bi kinetic mechanism. Nicotinic acid adenine dinucleotide, thionicotinamide adenine dinucleotide, and 3-aminopyridine adenine dinucleotide acted as inhibitors for this enzymatic crystallin. The inhibitors were competitive versus NAD+ and noncompetitive versus L-lactate. alpha-NAD+ was a noncompetitive inhibitor with respect to the usual beta-NAD+. D-Lactate, tartronate, and oxamate were strong dead-end inhibitors for the lactate dehydrogenase activity of epsilon-crystallin. Both D-lactate and tartronate were competitive inhibitors versus L-lactate while oxamate was a competitive inhibitor versus pyruvate. We conclude that the structural requirements for the substrate and coenzyme of epsilon-crystallin are similar to those of other dehydrogenases and that the carboxamide carbonyl group of the nicotinamide moiety is important for the coenzyme activity.     

A general method for relieving substrate inhibition in lactate dehydrogenases.

The mutation S163L in human heart lactate dehydrogenase removes substrate inhibition while only modestly reducing the turnover rate for pyruvate. Since this is the third enzyme to show this behaviour, we suggest that the S163L mutation is a general method for the removal of substrate inhibition in L-LDH enzymes. Engineering such enzymatic properties has clear industrial applications in the use of these enzymes to produce enantiomerically pure alpha-hydroxy acids. The mutation leads to two principal effects. (1) Substrate inhibition is caused by the formation of a covalent adduct between pyruvate and the oxidized form of the cofactor. The inability of S163L mutants to catalyse the formation of this inhibitory adduct is demonstrated. However, NMR experiments show that the orientation of the nicotinamide ring in the mutant NAD+ binary complex is not perturbed. (2) The mutation also leads to a large increase in the KM for pyruvate. The kinetic and binding properties of S163L LDH mutants are accounted for by a mechanism which invokes a non-productive, bound form of the cofactor. Molecular modelling suggests a structure for this non-productive enzyme-NADH complex.     

Conversion of inactive (phosphorylated) pyruvate dehydrogenase complex into active complex by the phosphate reaction in heart mitochondria is inhibited by alloxan-diabetes or starvation in the rat.

1. The conversion of inactive (phosphorylated) pyruvate dehydrogenase complex into active (dephosphorylated) complex by pyruvate dehydrogenase phosphate phosphatase is inhibited in heart mitochondria prepared from alloxan-diabetic or 48h-starved rats, in mitochondria prepared from acetate-perfused rat hearts and in mitochondria prepared from normal rat hearts incubated with respiratory substrates for 6 min (as compared with 1 min). 2. This conclusion is based on experiments with isolated intact mitochondria in which the pyruvate dehydrogenase kinase reaction was inhibited by pyruvate or ATP depletion (by using oligomycin and carbonyl cyanide m-chlorophenylhydrazone), and in experiments in which the rate of conversion of inactive complex into active complex by the phosphatase was measured in extracts of mitochondria. The inhibition of the phosphatase reaction was seen with constant concentrations of Ca2+ and Mg2+ (activators of the phosphatase). The phosphatase reaction in these mitochondrial extracts was not inhibited when an excess of exogenous pig heart pyruvate dehydrogenase phosphate was used as substrate. It is concluded that this inhibition is due to some factor(s) associated with the substrate (pyruvate dehydrogenase phosphate complex) and not to inhibition of the phosphatase as such. 3. This conclusion was verified by isolating pyruvate dehydrogenase phosphate complex, free of phosphatase, from hearts of control and diabetic rats an from heart mitochondria incubed for 1min (control) or 6min with respiratory substrates. The rates of re-activation of the inactive complexes were then measured with preparations of ox heart or rat heart phosphatase. The rates were lower (relative to controls) with inactive complex from hearts of diabetic rats or from heart mitochondria incubated for 6min with respiratory substrates. 4. The incorporation of 32Pi into inactive complex took 6min to complete in rat heart mitocondria. The extent of incorporation was consistent with three or four sites of phosphorylation in rat heart pyruvate dehydrogenase complex. 5. It is suggested that phosphorylation of sites additional to an inactivating site may inhibit the conversion of inactive complex into active complex by the phosphatase in heart mitochondria from alloxan-diabetic or 48h-starved rats or in mitochondria incubated for 6min with respiratory substrates.     

Zinc inhibition of pyruvate kinase of M-type isozyme

Inhibitory effect of Zn on the pyruvate kinase of M (muscle)-type isozyme was analyzed for the purpose of elucidating the cytotoxicity of Zn. Zn inhibited pyruvate kinase uncompetitively with respect to the substrate PEP, and competitively with respect to ADP. Quotient velocity plot calculated from the Zn-inhibition curves showed that Zn²⁺ as a ZnADP complex acted as competitive and uncompetitive inhibitors of the enzyme with respect to the substrate ADP and PEP, respectively: Zn²⁺ forms a ZnADP complex, which may bind to the ADP-binding site of the free enzyme with the K_i value of 1.4 μM causing competitive inhibition, or to the ADP-site of the enzyme-PEP complex with 2.6 μM resulting in uncompetitive inhibition. The inhibition of pyruvate kinase by Zn²⁺ may be responsible for the cytotoxicity of this metal by decreasing glycolytic flux.     

Pyruvate:NADP+ oxidoreductase from Euglena gracilis: the kinetic properties of the enzyme

The kinetic properties for the native forward reaction of pyruvate:NADP+ oxidoreductase from Euglena gracilis were determined. The substrate kinetics gave a pattern of a ping-pong mechanism involving a competitive substrate inhibition of CoA against pyruvate. The Km values for pyruvate, CoA, and NADP+ were estimated to be 27, 6.6, and 28 microM, respectively, and the Ki value of CoA against pyruvate was 28 microM. CO2 inhibited noncompetitively against pyruvate and NADP+, and uncompetitively against CoA. Acetyl-CoA showed a competitive inhibition with respect to pyruvate and an uncompetitive inhibition with respect to NADP+. NADPH inhibited competitively versus NADP+, noncompetitively versus CoA, and uncompetitively versus pyruvate. The kinetic behavior is consistent with a two-site ping-pong mechanism involving the substrate inhibition. From the kinetic mechanism, it is proposed that the enzyme has two catalytic sites linked by an intramolecular electron-transport chain. One of these is a thiamine pyrophosphate-containing catalytic site which reacts with pyruvate and CoA to form CO2 and acetyl-CoA, and the other site functions in the reduction of NADP+. In contrast, when methyl viologen was used as an artificial one-electron acceptor substituting for NADP+, the reaction gave a pattern characteristic of an octa uni ping-pong mechanism involving a competitive substrate inhibition of CoA against pyruvate.     

The conserved sequence NXX[S/T]HX[S/T]QDXXXT of the lactate/pyruvate:H(+) symporter subfamily defines the function of the substrate translocation pathway

In Saccharomyces cerevisiae Jen1p is a lactate/proton symporter belonging to the lactate/pyruvate:H(+) symporter subfamily (TC#2.A.1.12.2) of the Major Facilitator Superfamily. We investigated structure-function relationships of Jen1p using a rational mutational analysis based on the identification of conserved amino acid residues. In particular, we studied the conserved sequence (379)NXX[S/T]HX[S/T]QDXXXT(391). Substitution of amino acid residues N379, H383 or D387, even with very similar amino acids, resulted in a dramatic reduction of lactate and pyruvate uptake, but conserved measurable acetate transport. Acetate transport inhibition assays showed that these mutants conserve the ability to bind, but do not transport, lactate and pyruvate. More interestingly, the double mutation H383D/D387H, while behaving as a total loss-of-function allele for lactate and pyruvate uptake, can fully restore the kinetic parameters of Jen1p for acetate transport. Thus, residues N379, H383 or D387 affect both the transport capacity and the specificity of Jen1p. Substitutions of Q386 and T391 resulted in no or moderate changes in Jen1p transport capacities for lactate, pyruvate and acetate. On the other hand, Q386N reduces the binding affinities for all Jen1p substrates, while Q386A increases the affinity specifically for pyruvate. We also tested Jen1p specificity for a range of monocarboxylates. Several of the mutants studied showed altered inhibition constants for these acids. These results and 3D in silico modelling by homology threading suggest that the conserved motif analyzed is part of the substrate translocation pathway in the lactate/pyruvate:H(+) symporter subfamily.     

Kinetic analyses of the pyruvate dehydrogenase complex from Candida 107 (NCYC 911).

Candida 107 (NCYC 911) accumulates up to 45% of the biomass as triglycerides under conditions of nitrogenous substrate limitation in the medium. In oilseeds and adipocytes, lipid accumulation is preceded and accompanied by increased activity of key enzymes such as pyruvate dehydrogenase. However, in Candida 107, the activity of this complex was greatly reduced during lipogenesis. The initial velocity patterns were in accordance with a Hexa Uni Ping Pong mechanism. The Km values for the various substrates were similar to those found for the yeast Saccharomyces cerevisiae, but much higher than those reported for the mammalian enzyme. Product inhibition studies indicated that the Ki for acetyl coenzyme A and NADH were higher than those reported for other yeasts. The values for Ki were similar to those found for the liver enzyme, whereas the enzyme complex from heart had much lower Ki values for products. It has been suggested that in the heart and kidney, pyruvate dehydrogenase is regulated by product inhibition whereas in the liver this does not appear to be the mechanism. Therefore, it is probable, that like the liver enzyme, pyruvate dehydrogenase from Candida 107 may not be regulated by product inhibition.     

Kinetics of growth and substrate consumption of Escherichia coli ML 30 on two carbon sources.

When E. coli ML 30 is grown in batch culture on a mineral salt medium containing a mixed carbon source of glucose and pyruvate, there is no sequential utilization of the carbon sources. The consumption of glucose and pyruvate takes place simultaneously with reciprocal influence (inhibition) on rates of substrate uptake. The specific growth rate is greater than mupmax for pyruvate but smaller than musmax for glucose. In the paper three cases of kinetics of growth and of substrate consumption at several combinations of initial substrate concentrations are considered. A mathematical model is proposed and investigated. The model allows to describe the growth on glucose or on pyruvate not only as singular carbon sources, but also as a mixed carbon source with reciprocal inhibition on rates of substrate uptake. By data fitting parameters of growth and substrate consumption were found.     

The kinetic mechanism of pyruvate reduction by lactate dehydrogenase from Phycomyces blakesleeanus

The kinetics of pyruvate reduction by lactate dehydrogenase from Phycomyces blakesleeanus NRRL 1555 (-) have been determined at pH 6.0. Initial rate studies performed in the pyruvate reduction direction suggest that a sequential mechanism is operating. Product inhibition studies with NAD+ and L(+)-lactate are consistent with an ordered sequential mechanism if we considered that NAD+ mimics the NADH that binds cooperatively on the enzyme and also the existence of dead-end complex responsible for substrate inhibition by pyruvate at this pH value.     

Revisitation of the βCl-elimination reaction of D-amino acid oxidase: new interpretation of the reaction that sparked flavoprotein dehydrogenation mechanisms

D-amino acid oxidase (DAAO) from pig has been reported to catalyze the β-elimination of Cl(-) from βCl-D-alanine via abstraction of the substrate α-H as H(+) ("carbanion mechanism") (Walsh, C. T., Schonbrunn, A., and Abeles, R. H. (1971) J. Biol. Chem. 246, 6855-6866). In view of the fundamental mechanistic importance of this reaction and of the recent reinterpretation of the DAAO dehydrogenation step as occurring via a hydride mechanism, we reinvestigated the elimination reaction using yeast DAAO. That enzyme catalyzes the same reactions as the pig enzyme but with a much higher efficiency and a substantially different kinetic behavior. The reaction is initiated by a very rapid and fully reversible dehydrogenation step. This leads to an equilibrium (k(on) ≈ k(reverse)) between the complexes of oxidized enzyme-βCl-D-alanine and reduced enzyme-βCl-iminopyruvate. In the presence of O(2) the latter complex can partition between an oxidative half-reaction and elimination of Cl(-), which proceeds at a rate of ≈50 s(-1). This step forms a complex between oxidized enzyme and enamine that is characterized by a charge transfer absorption (which describes its rates of formation and decay). A minimal scheme that lists relevant steps of the reductive and oxidative half-reactions and elimination pathways along with the estimate of the corresponding rate constants is presented. β-Elimination of Cl(-) is proposed to originate at the locus of the enzyme-βCl-iminopyruvate complex. A chemical mechanism that can account for elimination is discussed in detail.     

Kinetic properties of the 2-oxoglutarate dehydrogenase complex from Azotobacter vinelandii evidence for the formation of a precatalytic complex with 2-oxoglutarate.

The 2-oxoglutarate dehydrogenase complex was purified from Azotobacter vinelandii. The complex consists of three components, 2-oxoglutarate dehydrogenase/decarboxylase (E1o), lipoate succinyltransferase (E2o) and lipoamide dehydrogenase (E3). Upon purification, the E3 component dissociates partially from the complex. From reconstitution experiments, the Kd for E3 was found to be 26 nM, about 30 times higher than that for the pyruvate dehydrogenase complex. The Km values for the substrates 2-oxoglutarate, CoA and NAD+ were found to be 0.15, 0.014 and 0.17 mM, respectively. The system has a high specificity for 2-oxoglutarate, which is determined by the action of both E1o and E2o. Above 4 mM substrate inhibition is observed. From steady-state inhibition experiments with substrate analogs, two substrate-binding modes are revealed at different degrees of saturation of the enzyme with 2-oxoglutarate. At low substrate concentrations (10(-6) to 10(-5) M), the binding mainly depends on the interaction of the enzyme with the substrate carboxyl groups. At a higher degree of substrate saturation (10(-4) to 10(-3) M) the relative contribution of the 2-oxo group in the binding increases. A kinetic analysis points to a single binding site for a substrate analog under steady state conditions. Saturation of this site with an analog indicates that two kinetically different complexes are formed with 2-oxoglutarate in the course of catalysis. From competition studies with analogs it is concluded that one of these complexes is formed at the site that is sterically identical to the substrate inhibition site. The data obtained are represented by a minimal scheme that considers formation of a precatalytic complex SE between the substrate and E1o before the catalytic complex ES, in which the substrate is added to the thiamin diphosphate cofactor, is formed. The incorrect orientation of the substrate molecule in SE or the occupation of this site by analogs is supposed to cause substrate or analog inhibition, respectively.     

Cooperative interaction of pyruvate dehydrogenase from pigeon breast muscle]

Cooperative interaction of pyruvate with the pyruvate dehydrogenase (PD) complex from pigeon breast muscle was shown. The sigmoidal dependence of the reaction rate on pyruvate concentration was observed for the PD complex. The Hill coefficient is equal to 1,5; no inhibition by the substrate (up to 2.2.10(-3) M) was found. The kinetic behaviour of the isolated pyruvate dehydrogenase component (PDH) analyzed under similar conditions, is more complex; this may be probably due to the presence of oligomeric forms with different molecular weights and specific activities. The competitive inhibitor of the PD complex--an amide of pyruvic acid (PA) (Ki=6.3-10(-6) M) activates the enzyme at low concentrations (less than 2,10(-6) M). When PA is present, the dependence of the reaction rate on pyruvate concentration gives a usual hyperbolic curve, v of [S]o. It is concluded that pyruvate may have a regulatory effect on the activity of muscle PD complex.     

Protein-protein interactions and substrate channeling in orthologous and chimeric aldolase-dehydrogenase complexes

Bacterial aldolase-dehydrogenase complexes catalyze the last steps in the meta cleavage pathway of aromatic hydrocarbon degradation. The aldolase (TTHB246) and dehydrogenase (TTHB247) from Thermus thermophilus were separately expressed and purified from recombinant Escherichia coli. The aldolase forms a dimer, while the dehydrogenase is a monomer; these enzymes can form a stable tetrameric complex in vitro, consisting of two aldolase and two dehydrogenase subunits. Upon complex formation, the K(m) value of 4-hydroxy-2-oxopentanoate, the substrate of TTHB246, is decreased 4-fold while the K(m) of acetaldehyde, the substrate of TTHB247, is increased 3-fold. The k(cat) values of each enzyme were reduced by ~2-fold when they were in a complex. The half-life of TTHB247 at 50 °C increased by ~4-fold when it was in a complex with TTHB246. The acetaldehyde product from TTHB246 could be efficiently channelled directly to TTHB247, but the channeling efficiency for the larger propionaldehyde was ~40% lower. A single A324G substitution in TTHB246 increased the channeling efficiency of propionaldehyde to a value comparable to that of acetaldehyde. Stable and catalytically competent chimeric complexes could be formed between the T. thermophilus enzymes and the orthologous aldolase (BphI) and dehydrogenase (BphJ) from the biphenyl degradation pathway of Burkholderia xenovorans LB400. However, channeling efficiencies for acetaldehyde in these chimeric complexes were ~10%. Structural and sequence analysis suggests that interacting residues in the interface of the aldolase-dehydrogenase complex are highly conserved among homologues, but coevolution of partner enzymes is required to fine-tune this interaction to allow for efficient substrate channeling.     

Isolation of BsoBI restriction endonuclease variants with altered substrate specificity

BsoBI is a thermophilic restriction endonuclease that cleaves the degenerate DNA sequence C/PyCGPuG (where/=the cleavage site and Py=C or T, Pu=A or G). In the BsoBI-DNA co-crystal structure the D246 residue makes a water-mediated hydrogen bond to N6 of the degenerate base adenine and was proposed to make a complementary bond to O6 of the alternative guanine residue. To investigate the substrate specificity conferred by D246 and to potentially alter BsoBI specificity, the D246 residue was changed to the other 19 amino acids. Variants D246A, D246C, D246E, D246R, D246S, D246T, and D246Y were purified and their cleavage activity determined. Variants D246A, D246S, and D246T display 0.2% to 0.7% of the wild-type cleavage activity. However, the substrate specificity of the three variants is altered significantly. D246A, D246S, and D246T cleave CTCGAG sites poorly. In filter binding assays using oligonucleotides, wild-type BsoBI shows almost equal affinity for CTCGAG and CCCGGG sites. In contrast, the D246A variant shows 70-fold greater binding affinity for the CCCGGG substrate. Recycled mutagenesis was carried out on the D246A variant, and revertants with enhanced activity were isolated by their dark blue phenotype on a dinD Colon, two colons lacZ DNA damage indicator strain. Most of the amino acid substitutions present within the revertants were located outside the DNA-protein interface. This study demonstrates that endonuclease mutants with altered specificity and non-lethal activity can be evolved towards more active variants using a laboratory evolution strategy.     

The reaction of pyruvate with saccharopine dehydrogenase.

The preceding paper in this journal has reported that pyruvate could be substituted for 2-oxo-glutarate as a substrate of saccharopine dehydrogenase [epsilon-N-(L-glutaryl-2)-L-lysine:NAD oxidoreductase (L-lysine-forming) in the direction of reductive condensation. In the present communication, the kinetic mechanism of saccharopine dehydrogenase reaction with NADH, L-lysine and pyruvate as reactants is reported. The results of initial velocity study, inhibition studies with lysine analogs and a reaction product, NAD+, are consistent with an ordered mechanism with the coenzyme binding first and pyruvate last. The reaction mechanism is at variance with that of the normal reaction in which 2-oxoglutarate is the substrate, in that the order of addition of the amino and oxo acid substrates is reversed. This fact suggests that there exists a small degree of randomness in the binding of amino and oxo acid substrates. From a product inhibition study, NAD+ was shown to be the last reactant released. Saccharopine [epsilon-N-(L-glutaryl-2)-L-lysine] was found to act as a potent dead-end inhibitor of the condensation reactions (of lysine and 2-oxoglutarate, and of lysine and pyruvate) by forming an abortive E. NADH. saccharopine complex.     

The regulation of pyruvate dehydrogenase by fatty acids in isolated rabbit heart mitochondria.

The rate of pyruvate oxidation by isolated rabbit heart mitochondria was inhibited by fatty acylcarnitine derivatives. The extent of inhibition by pyruvate oxidation in State 3 was greatest with palmitylcarnitine and only a minimal inhibition was observed with acetylcarnitine, while octanoylcarnitine or octanoate caused an intermediate extent of inhibition. Analyses of the intramitochondrial $\text{ATP}\text{ADP}$ and $\text{NADH}\text{NAD}{}^{\mathrm{+}}$ ratios under the different conditions of incubation indicated that it is unlikely that changes in either or both of these parameters were the primary negative effectors of the rate of pyruvate oxidation. A positive correlation between the decrease in the rate of pyruvate oxidation and the decrease in the level of free CoASH in the mitochondria was observed. Extraction and assay of the pyruvate dehydrogenase from rabbit heart mitochondria during the time course of the fatty acid-mediated inhibition of pyruvate oxidation indicated that pyruvate dehydrogenase was strongly inactivated when palmitylcarnitine was the fatty acid, while incubation with octanoate and acetylcarnitine resulted in less extensive inactivation of pyruvate dehydrogenase. Measurement of the effects of NADH, NAD⁺, acetyl-CoA, and CoASH on the inactivation of pyruvate dehydrogenase extracted from rabbit heart mitochondria indicated that NADH and acetyl-CoA activated the pyruvate dehydrogenasee kinase while CoASH strongly inhibited the kinase and NAD⁺ was without effect. In addition, palmityl-CoA and octanoyl-CoA had little, if any, effect on the pyruvate dehydrogenase kinase activity. It was observed that palmityl-CoA but not octanoyl-CoA strongly inhibited the activity of the extracted pyruvate dehydrogenase. Hence, it is concluded that (a) decreased mitochondrial CoASH levels, which essentially remove a potent inhibitor of the pyruvate dehydrogenase kinase, (b) possibly a diminished free CoASH supply, which may be utilized as a substrate for the active complex, and (c) direct inhibitory effects of palmityl-CoA on the active form of the pyruvate dehydrogenase complex combine to make palmitylcarnitine a much more potent inhibitor of mitochondrial pyruvate oxidation than shorter chain length acylcarnitine derivatives.