X-ray structures of Aerococcus viridans lactate oxidase and its complex with D-lactate at pH 4.5 show an alpha-hydroxyacid oxidation mechanism

l-Lactate oxidase (LOX) belongs to a family of flavin mononucleotide (FMN)-dependent α-hydroxy acid-oxidizing enzymes. Previously, the crystal structure of LOX (pH 8.0) from Aerococcus viridans was solved, revealing that the active site residues are located around the FMN. Here, we solved the crystal structures of the same enzyme at pH 4.5 and its complex with d-lactate at pH 4.5, in an attempt to analyze the intermediate steps. In the complex structure, the d-lactate resides in the substrate-binding site, but interestingly, an active site base, His265, flips far away from the d-lactate, as compared with its conformation in the unbound state at pH 8.0. This movement probably results from the protonation of His265 during the crystallization at pH 4.5, because the same flip is observed in the structure of the unbound state at pH 4.5. Thus, the present structure appears to mimic an intermediate after His265 abstracts a proton from the substrate. The flip of His265 triggers a large structural rearrangement, creating a new hydrogen bonding network between His265-Asp174-Lys221 and, furthermore, brings molecular oxygen in between d-lactate and His265. This mimic of the ternary complex intermediate enzyme-substrate-O₂ could explain the reductive half-reaction mechanism to release pyruvate through hydride transfer. In the mechanism of the subsequent oxidative half-reaction, His265 flips back, pushing molecular oxygen into the substrate-binding site as the second substrate, and the reverse reaction takes place to produce hydrogen peroxide. During the reaction, the flip-flop action of His265 has a dual role as an active base/acid to define the major chemical steps. Our proposed reaction mechanism appears to be a common mechanistic strategy for this family of enzymes.     

Properties and mechanism of action of creatine kinase from ox smooth muscle. Anion effects compared with pyruvate kinase

1. An improved purification procedure for the brain-type creatine kinase from ox smooth muscle is described. 2. Michaelis constants show the characteristic dependence on the concentration of the second substrate: the derived constants are compared with those for the enzyme from ox brain. 3. Inhibition by iodoacetamide gives a biphasic curve and the total extent of the reaction depends on the enzyme concentration. The rate of inhibition at pH8.6 is not affected by creatine plus MgADP or by a range of simple anions. Addition of creatine plus MgADP plus either NO(3) (-) or Cl(-) ions affords 71.5 and 44% protection respectively. ADP could be replaced by 2-deoxy-ADP but not by alphabeta-methylene ADP, XDP, IDP, GDP or CDP. Nucleotides that did not protect would not act as substrates. 4. Difference-spectra measurements support the interpretation that addition of NO(3) (-) ions to the enzyme-creatine-MgADP complex causes further conformational changes in the enzyme accompanying the formation of a stable quaternary enzyme-creatine-NO(3) (-)-MgADP complex that simulates an intermediate stage in the transphosphorylation reaction. However, the enzyme structure is partially destabilized by quaternary-complex formation. IDP apparently fails to act as a substrate because it cannot induce the necessary conformational change. This behaviour is compared with that of rabbit skeletal muscle creatine kinase. 5. With pyruvate kinase from rabbit muscle, anions activate in the absence of an activating cation and either inhibit or have no effect in its presence. 6. Both activation and inhibition were competitive with respect to the substrate, phosphoenolpyruvate, and curved double-reciprocal plots were obtained. The results may be interpreted in terms of co-operatively induced conformational changes, and this is supported by difference-spectra measurements. However, the Hill coefficient of 1 was not significantly altered. 7. Inhibition by lactate plus pyruvate is less than additive, indicating that both bind to the same site on the enzyme, whereas that by lactate plus NO(3) (-) is additive, indicating binding at separate sites. It is inferred that a quaternary enzyme-pyruvate-NO(3) (-)-MgADP complex could form, but no evidence was obtained to suggest that it possessed special properties comparable with those found with creatine kinase. The implications of these findings for the unidirectional nature of the mechanism of pyruvate kinase is discussed. 8. Lactate or alpha-hydroxybutyrate could not act instead of pyruvate to form a stable quaternary complex, although both activate the K(+)-free enzyme. Only the former inhibits the K(+)-activated enzyme. The activating cation both lowers the Michaelis constant for phosphoenolpyruvate and tightens up the specificity of its binding site.     

Lipoic acid residues in a take-over mechanism for the pyruvate dehydrogenase multienzyme complex of Escherichia coli.

The pyruvate dehydrogenase complex of Escherichia coli contains two lipoic acid residues per dihydrolipoamide acetyltransferase chain, and these are known to engage in the part-reactions of the enzyme. The enzyme complex was treated with trypsin at pH 7.0, and a partly proteolysed complex was obtained that had lost almost 60% of its lipoic acid residues although it retained 80% of its pyruvate dehydrogenase-complex activity. When this complex was treated with N-ethylmaleimide in the presence of pyruvate and the absence of CoASH, the rate of modification of the remaining S-acetyldihydrolipoic acid residues was approximately equal to the accompanying rate of loss of enzymic activity. This is in contrast with the native pyruvate dehydrogenase complex, where under the same conditions modification proceeds appreciably faster than the loss of enzymic activity. The native pyruvate dehydrogenase complex was also treated with lipoamidase prepared from Streptococcus faecalis. The release of lipoic acid from the complex followed zero-order kinetics for most of the reaction, whereas the accompanying loss of pyruvate dehydrogenase-complex activity lagged substantially behind. These results eliminate a model for the enzyme mechanism in which specifically one of the two lipoic acid residues on each dihydrolipoamide acetyltransferase chain is essential for the reaction. They are consistent with a model in which the dihydrolipoamide acetyltransferase component contains more lipoic acid residues than are required to serve the pyruvate decarboxylase subunits under conditions of saturating substrates, enabling the function of an excised or inactivated lipoic acid residue to be taken over by another one. Unusual structural properties of the enzyme complex might permit this novel feature of the enzyme mechanism.     

Enzymes related to lactate metabolism in green algae and lower land plants

Cell-free extracts of Chlorella pyrenoidosa contained two enzymes capable of oxidizing d-lactate; these were glycolate dehydrogenase and NAD(+)-dependent d-lactate dehydrogenase. The two enzymes could be distinguished by differential centrifugation, glycolate dehydrogenase being largely particulate and NAD(+)-d-lactate dehydrogenase being soluble. The reduction of pyruvate by NADH proceeded more rapidly than the reverse reaction, and the apparent Michaelis constants for pyruvate and NADH were lower than for d-lactate and NAD(+). These data indicated that under physiological conditions, the NAD(+)-linked d-lactate dehydrogenase probably functions to produce d-lactate from pyruvate.Lactate dehydrogenase activity dependent on NAD(+) was found in a number of other green algae and in the green tissues of a few lower land plants. When present in species which contain glycolate oxidase rather than glycolate dehydrogenase, the enzyme was specific for l-lactate rather than d-lactate. A cyclic system revolving around the production and utilization of d-lactate in some species and l-lactate in certain others is proposed.     

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.     

The elementary reactions of the pig heart pyruvate dehydrogenase complex. A study of the inhibition by phosphorylation.

1. A method was devised for preparing pig heart pyruvate dehydrogenase free of thiamin pyrophosphate (TPP), permitting studies of the binding of [35S]TPP to pyruvate dehydrogenase and pyruvate dehydrogenase phosphate. The Kd of TPP for pyruvate dehydrogenase was in the range 6.2-8.2 muM, whereas that for pyruvate dehydrogenase phosphate was approximately 15 muM; both forms of the complex contained about the same total number of binding sites (500 pmol/unit of enzyme). EDTA completely inhibited binding of TPP; sodium pyrophosphate, adenylyl imidodiphosphate and GTP, which are inhibitors (competitive with TPP) of the overall pyruvate dehydrogenase reaction, did not appreciably affect TPP binding. 2. Initial-velocity patterns of the overall pyruvate dehydrogenase reaction obtained with varying TPP, CoA and NAD+ concentrations at a fixed pyruvate concentration were consistent with a sequential three-site Ping Pong mechanism; in the presence of oxaloacetate and citrate synthase to remove acetyl-CoA (an inhibitor of the overall reaction) the values of Km for NAD+ and CoA were 53+/- 5 muM and 1.9+/-0.2 muM respectively. Initial-velocity patterns observed with varying TPP concentrations at various fixed concentrations of pyruvate were indicative of either a compulsory order of addition of substrates to form a ternary complex (pyruvate-Enz-TPP) or a random-sequence mechanism in which interconversion of ternary intermediates is rate-limiting; values of Km for pyruvate and TPP were 25+/-4 muM and 50+/-10 nM respectively. The Kia-TPP (the dissociation constant for Enz-TPP complex calculated from kinetic plots) was close to the value of Kd-TPP (determined by direct binding studies). 3. Inhibition of the overall pyruvate dehydrogenase reaction by pyrophosphate was mixed non-competitive versus pyruvate and competitive versus TPP; however, pyrophosphate did not alter the calculated value for Kia-TPP, consistent with the lack of effect of pyrophosphate on the Kd for TPP. 4. Pyruvate dehydrogenase catalysed a TPP-dependent production of 14CO2 from [1-14C]pyruvate in the absence of NAD+ and CoA at approximately 0.35% of the overall reaction rate; this was substantially inhibited by phosphorylation of the enzyme both in the presence and absence of acetaldehyde (which stimulates the rate of 14CO2 production two- or three-fold). 5. Pyruvate dehydrogenase catalysed a partial back-reaction in the presence of TPP, acetyl-CoA and NADH. The Km for TPP was 4.1+/-0.5 muM. The partial back-reaction was stimulated by acetaldehyde, inhibited by pyrophosphate and abolished by phosphorylation. 6. Formation of enzyme-bound [14C]acetylhydrolipoate from [3-14C]pyruvate but not from [1-14C]acetyl-CoA was inhibited by phosphorylation. Phosphorylation also substantially inhibited the transfer of [14C]acetyl groups from enzyme-bound [14C]acetylhydrolipoate to TPP in the presence of NADH. 7...     

Purification and characterization of a highly unusual tetrameric D-lactate dehydrogenase from the muscle of the giant barnacle, Balanus nubilus Darwin

d-Lactate dehydrogenase from the depressor muscle of the giant barnacle, Balanus nubilus Darwin, was purified to homogeneity. The molecular weight of this enzyme, as judged by meniscus depletion sedimentation equilibrium and gel filtration, corresponds to a tetrameric subunit organization unlike the d-lactate dehydrogenases from the horeseshoe crab, Limulus polyphemus, and the polychaete, Nereis virens, which are dimeric. It is concluded that substrate stereospecificity and the degree of subunit organization are two independent parameters in the evolution of lactate dehydrogenases. The amino acid composition of B. nubilusd-lactate dehydrogenase shows general similarities to both the Limulus enzyme and the l-lactate dehydrogenase from the lobster, Homarus americanus, except for an unusually high cysteine content (10 residues per subunit). The isoelectric point of the barnacle enzyme is 5.0. B. nubilusd-lactate dehydrogenase is clearly a muscle-type enzyme, as it displays very little substrate inhibition at high pyruvate concentrations. The catalytic properties of this enzyme, including high reactivity with α-ketobutyrate and α-hydroxybutyrate, lowered pH optimum (7.5) for lactate oxidation, and relative insensitivity to oxamate, also set it apart from other animal d-lactate dehydrogenases.     

Two separate pathways for d-lactate oxidation by Saccharomyces cerevisiae mitochondria which differ in energy production and carrier involvement

In this work we looked at whether and how mitochondria isolated from Saccharomyces cerevisiae (SCM) oxidize d-lactate. We found that: (1). externally added d-lactate causes oxygen uptake by SCM with P/O ratio equal to 1.5; in the presence of antimycin A (AA), P/O ratio was 1.8, differently in the presence of the non-penetrant alpha-cyanocinnamate (alpha-CCN-) no P/O ratio could be measured. Consistently, mitochondrial electrical membrane potential (deltapsi) generation was found, due to externally added d-lactate in the presence of antimycin A, but not of alpha-CCN-. (2). SCM oxidize d-lactate in two different manners: (i). via inner membrane d-lactate dehydrogenase which leads to d-lactate oxidation without driving deltapsi generation and ATP synthesis and (ii). via the matrix d-lactate dehydrogenase, which drives deltapsi generation and ATP synthesis by using taken up d-lactate. (3). Pyruvate newly synthesised in the mitochondrial matrix is exported via the novel d-lactate/pyruvate antiporter. d-Lactate/pyruvate antiport proved to regulate the rate of pyruvate efflux in vitro. (4). The existence of the d-lactate/H+ symporter is also proposed as shown by mitochondrial swelling. The d-lactate carriers and d-lactate dehydrogenases could account for the removal of the toxic methylglyoxal from cytosol, as well as for the d-lactate-dependent gluconeogenesis.     

Kinetics and mechanism of acetohydroxy acid synthase isozyme III from Escherichia coli

Acetohydroxy acid synthase (AHAS, EC 4.1.3.18) isozyme III from Escherichia coli has been studied in steady-state kinetic experiments in which the rates of formation of acetolactate (AL) and acetohydroxybutyrate (AHB) have been determined simultaneously. The ratio between the rates of production of the two alternative products and the concentrations of the substrates pyruvate and 2-ketobutyrate (2KB) leading to them, R, VAHB/VAL = R[( 2KB]/[pyruvate]), was found to be 40 +/- 3 under a wide variety of conditions. Because pyruvate is a common substrate in the reactions leading to both products and competes with 2-ketobutyrate to determine whether AL or AHB is formed, steady-state kinetic studies are unusually informative for this enzyme. At a given pyruvate concentration, the sum of the rates of formation of AL and AHB was nearly independent of the 2-ketobutyrate concentration. On the basis of these results, a mechanism is proposed for the enzyme that involves irreversible and rate-determining reaction of pyruvate, at a site which accepts 2-ketobutyrate poorly, if at all, to form an intermediate common to all the reactions. In the second phase of the reaction, various 2-keto acids can compete for this intermediate to form the respective acetohydroxy acids. 2-Keto acids other than the natural substrates pyruvate and 2-ketobutyrate may also compete, to a greater or lesser extent, in the second phase of the reaction to yield alternative products, e.g., 2-ketovalerate is preferred by about 2.5-fold over pyruvate. However, the presence of an additional keto acid does not affect the relative specificity of the enzyme for pyruvate and 2-ketobutyrate; this further supports the proposed mechanism. The substrate specificity in the second phase is an intrinsic property of the enzyme, unaffected by pH or feedback inhibitors.(ABSTRACT TRUNCATED AT 250 WORDS)     

The NADH dehydrogenase of the respiratory chain of Escherichia coli. II. Kinetics of the purified enzyme and the effects of antibodies elicited against it on membrane-bound and free enzyme.

The purified respiratory chain NADH dehydrogenase of Escherichia coli oxidizes NADH with either dichlorophenolindophenol (DCIP). ferricyanide, or menadione as electron acceptors, with values for NADH are similar with the three electron acceptors (approximately 50 muM). The purified enzyme contains no flavin and has an absolute requirement for FAD, with Km values around 4 muM. The pH optimum of the enzyme appears to be between 6.5 and 7; the optimum is difficult to establish because of nonenzymatic reduction of DCIP at the lower pH values. Potassium cyanide stimulates the DCIP reductase activity about 2-fold, but has no effect on ferricyanide reductase. The enzyme exhibits hyperbolic kinetics with respect to NADH concentration in both the ferricyanide and DCIP reductase assays, but cooperatively is seen in the menadione reductase reaction. NAD+ is an effective competitive inhibitor of the reaction (Ki congruent to 20 muM); in the presence of NAD+, the NADH saturation curve becomes cooperative, even in the DCIP reductase assay. Many adenine containing nucleotides are competitive inhibitors of the enzyme. The apparent Ki values for these nucleotides as inhibitors of the purified enzyme, the membrane-bound NADH dehydrogenase, and the NADH oxidase are equivalent. An examination of inhibitory effects of a series of adenine nucleotides suggests that the inhibitors act as analogues of NAD+, which is the true physiological inhibitor. The results suggest that the enzyme in situ is always partially inhibited by the levels of NAD- in the E coli cell, and thus behaves in a cooperative fashion to changes in the NAD+/NADH ratio. An antibody has been elicited against the purified NADH dehydrogenase. Immunodiffusion and crossed immunoelectrophoresis show that the antibody is directed principally against the NADH dehydrogenase, with some activity against minor contaminants in the purified preparation. The antibody inhibits NADH dehydrogenase activity 50% at saturating levels. When this antibody preparation is used to examine solubilized membrane preparations, two major immunoprecipitates are found. A parallel inhibition of the membrane-bound NADH dehydrogenase and NADH oxidase activities is seen, supporting the hypothesis that the purified enzyme is indeed a component of the respiratory chain-dependent NADH oxidase pathway.     

Kinetic mechanism of phosphoenolpyruvate carboxykinase (GTP) from rat liver cytosol. Product inhibition, isotope exchange at equilibrium, and partial reactions

Initial velocity studies of rat liver cytosolic P-enolpyruvate carboxykinase in the direction of P-enolpyruvate formation gave intersecting double reciprocal plots indicating that the reaction conforms to a sequential reaction pathway. A complete product inhibition study with MnGDP-, P-enolpyruvate, and HCO3- as product inhibitors indicated that all patterns were noncompetitive. Isotope exchange at equilibrium with exchange between the substrate/product pairs GTP/GDP oxalacetate/HCO3-, and oxalacetate/P-enolpyruvate while varying the concentration of substrate/product pairs in fixed constant ratio gave no complete inhibitory patterns as the concentration of the constant ratio pairs approached saturation. The exchange rates between the substrate/product pairs differed by a factor of 40 when compared under the same assay conditions. These results were interpreted in terms of a random reaction mechanism in which true dead-end complexes do not form and in which the rate-limiting step is not the interconversion of the ternary quarternary central complexes. In addition to the formation of P-enolpyruvate from oxalacetate and MnGTP2-, the enzyme catalyzes the decarboxylation of oxalacetate to pyruvate in the absence of MnGTP2-. This reaction occurs only slowly in the absence of GDP and most rapidly in the presence of MnGDP-. When only MnGTP2- and oxalacetate are present, no pyruvate is formed, and oxalacetate is converted stoichiometrically to P-enolpyruvate. The enzyme also catalyzes the exchange of [14C]GDP into GTP in the absence of P-enolpyruvate. This exchange is stimulated by the presence of HCO3-. When enzyme is incubated with MnGTP2- in the presence or absence of HCO3-, there is no hydrolysis to form GDP and P1. The two partial reactions, namely the exchange of [14C]GDP with the E.HCO3.MnGTP or E.MnGTP complex and the formation of pyruvate from the E.oxalacetate.MnGDP complex provide pathways by which the expected dead-end complexes can be converted to enzyme forms which can return to the catalytic or exchange sequence.     

Effects of magnesium, manganese and adenosine triphosphate ions on pyruvate carboxylase from baker''s yeast

1. Pyruvate carboxylase from baker's yeast acts with either MgATP(2-) or MnATP(2-) as substrate. The optimum pH for the enzyme reaction is 8.0 with MgATP(2-) and 7.0 with MnATP(2-). 2. When the reaction velocity is plotted against MgATP(2-) (or MnATP(2-)) concentration slightly sigmoid curves are obtained, either in the presence or in the absence of acetyl-CoA (an allosteric activator). In the presence of excess of free Mg(2+) (or Mn(2+)) the curves turn into hyperbolae, whereas in the presence of excess of free ATP(4-) the apparent sigmoidicity of the curves increases. 3. The sigmoidicity of the plots of v against MgATP(2-) (or MnATP(2-)) concentration can be explained by the inhibitory effect of free ATP(4-), the concentration of which, in the experimental conditions employed, is significant and varies according to the total concentration of the ATP-magnesium chloride (or ATP-manganese chloride) mixture. Free ATP(4-) behaves as a negative modifier of yeast pyruvate carboxylase. 4. The effect of high concentrations of Mg(2+) (or Mn(2+)) on the kinetics of yeast pyruvate carboxylase can be explained as a deinhibition with respect to ATP(4-), instead of a direct enzyme activation. 5. At pH6.5 manganese chloride is more effective than magnesium chloride as enzyme activator even in the presence of a great excess (16-fold) of the latter. This is consistent with a significant contribution of the MnATP(2-) complex to the activity of yeast pyruvate carboxylase, in medium conditions resembling those existing inside the yeast cell (pH6.25-6.75; 12mm-magnesium chloride and 0.75mm-manganese chloride). 6. The physiological significance of the enzyme inhibition by free ATP(4-) is doubtful since the Mg(2+) and Mn(2+) concentrations reported to exist inside the yeast cell are sufficient to decrease ATP(4-) concentrations to ineffective values.     

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.     

The role of biotin and sodium in the decarboxylation of oxaloacetate by the membrane-bound oxaloacetate decarboxylase from Klebsiella aerogenes

The biotin-containing oxaloacetate decarboxylase from Klebsiella aerogenes catalyzed the Na+-dependent decarboxylation of oxaloacetate to pyruvate and bicarbonate (or CO2) but not the reversal of this reaction, not even in the presence of an oxaloacetate trapping system. The enzyme catalyzed an avidin-sensitive isotopic exchange between [1-14C]pyruvate and oxaloacetate, which indicated the intermediate formation of a carboxybiotin enzyme. Sodium ions were not required for this partial reaction, but promoted the second partial reaction, the decarboxylation of the carboxybiotin enzyme, thus accounting for the Na+ requirement of the overall reaction. Therefore, the 14CO2-enzyme which was formed upon incubation of the decarboxylase with [4-15C]oxaloacetate, could only be isolated if Na+ ions were excluded. Preincubation of the decarboxylase with avidin also prevented its labelling with 14CO2. The isolated 14CO2-labelled oxaloacetate decarboxylase revealed the following properties. It was slowly decarboxylated at neutral pH and rapidly upon acidification. The 14CO2 residues of the 14CO2-enzyme could be transferred to pyruvate yielding [4-14C]oxaloacetate. In the presence of Na+ this 14CO2 transfer was repressed by the simultaneous decarboxylation of the 14CO2-enzyme. However, Na+ alone was insufficient as a cofactor for the decarboxylation of the isolated 14CO2-enzyme, since this required pyruvate in addition to Na+. It is therefore concluded that the decarboxylation of oxaloacetate proceeds over a CO2-enzyme--pyruvate complex and that free CO2-enzyme is an abortive reaction intermediate. The activation energy of the enzymic decarboxylation of oxaloacetate changed with temperature and was about 113 kJ below 11 degrees C, 60 kJ between 11 degrees C and 31 degrees C and 36 kJ between 31--45 degrees C.     

Decarboxylation of oxalacetate by pyruvate carboxylase

The decarboxylation of oxalacetate by pyruvate carboxylase in the absence of ADP and Pi is stimulated 400-fold by the presence of oxamate, which is an inhibitory analogue of pyruvate. The observation of substrate inhibition when either oxamate or oxalacetate is varied at a fixed concentration of the other indicates that both molecules bind at the same site on the enzyme. The pH profiles for this reaction show no evidence of the involvement of an enzymic acid-base catalyst, suggesting that the proton and CO2 units may be exchanged directly between the reactants (although CO2 sequestered in the active site may be an intermediate in the process). The pH profiles of the full reverse reaction of pyruvate carboxylase in which oxalacetate decarboxylation is coupled to ATP formation and where Pi is the variable substrate do, however, indicate that such an acid-base catalyst is involved in the other partial reaction of the enzyme in proton transfer to and from biotin. The enzyme also displays two oxamate-independent oxalacetate decarboxylating activities, one of which is biotin-dependent and the other is independent of biotin.     

Studies on the cyclic 3':5'-AMP-stimulated pig liver protein kinase reaction with pyruvate kinase as substrate.

The phosphorylation of pig liver pyruvate kinase by cyclic adenosine 3':5'-monophosphate-dependent protein kinase has been studied. For comparison, mixed histone and a synthetic heptapeptide were also used as substrates. Protein kinase was purified by chromatography on DEAE-cellulose, hydroxyapatite, and Sephadex G-200. The enzyme was stimulated by cyclic AMP with apparent Ka values of 2.5 and 0.8 x 10-7 M for pyruvate kinase and histone substrates, respectively. Divalent cations were essential for the activity of the protein kinase. Variation of the concentration of ATP resulted in approximately straight lines in Lineweaver-Burk plots for the phosphorylation of both pyruvate kinase and mixed histone. The apparent Km values for ATP were 21 and 11 muM, respectively. The phosphorylation rate increased with the concentration of pyruvate kinase even at a concentration of 2 muM pyruvate kinase. At a high ionic strength, the phosphorylation rate of both pyruvate kinase and histone decreased. The phosphorylation rate varied markedly with pH in imidazole/HC1 and Tris/HC1 buffers. At slightly alkaline pH values, pyruvate kinase was phosphorylated at a much higher rate than pH7, but this was not the case for histone. At pH 8.5, the phosphorylation rate of pyruvate kinase was 3.5 times the rate at pH 7, while the corresponding increase for the histone phosphorylation was 50 per cent. In potassium phosphate buffers, the phosphorylation rate of both substrates did not change significantly over the pH range studied. Arrhenius' plots of the protein kinase reaction resulted in a break at about 10 degrees when pyruvate kinase was used as substrate, whereas a straight line was obtained when using histone. The negative allosteric effectors of pyruvate kinase, alanine, and phenylalanine, increased the phosphorylation rate of pyruvate kinase at pH 8 by 50 and 120 per cent, respectively. The same effectors did not influence the phosphorylation rate of mixed histone or a synthetic heptapeptide. It is concluded that the conformations adopted by pyruvate kinase in the presence of allosteric inhibitors make it a better substrate for the protein kinase.     

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.     

Purification and characterization of N-methylalanine dehydrogenase.

Cell free extracts of Pseudomonas MS previously have been shown to carry out the synthesis of a novel amino acid, N-methylalanine (Kung, H.F., and Wagner, C. (1970) Biochim. Biophys. Acta 201, 513-516). An enzyme has been isolated from this organism which is responsible for the synthesis of N-methylalanine. The stoichiometry of the reaction catalyzed by this enzyme leads to the following formulation: Methylamine + pyruvate + NADPH + H-+ yields N-methylalanine + NADP-+ + H2O. This enzyme has been physically separated from alanine dehydrogenase, which is also present in these extracts. This new enzyme has been named N-methylalanine dehydrogenase. It has been purified to near homogeneity as judged by disc gel electrophoresis. Gel filtration chromatography showed that N-methylalanine dehydrogenase has an apparent molecular weight of 77,000, while electrophoresis in sodium dodecyl sulfate gave rise to a single band with a molecular weight of approximately 36,500. The enzyme is optimally active in the pH range between 8.2 and 8.6. The apparent K-m values for pyruvate, NADPH, and methylamine, respectively, are 1-5 times 10 minus 2 M, 3-5 times 10 minus 5 M, and 7.5 times 10 minus 2 M.     

Thermostable alanine dehydrogenase from thermophilic Bacillus sphaericus DSM 462. Purification, characterization and kinetic mechanism

Alanine dehydrogenase (L-alanine: NAD+ oxidoreductase, deaminating) was simply purified to homogeneity from a thermophile, Bacillus sphaericus DSM 462, by ammonium sulfate fractionation, red-Sepharose 4B chromatography and preparative slab gel electrophoresis. The enzyme had a molecular mass of about 230 kDa and consisted of six subunits with an identical molecular mass of 38 kDa. The enzyme was much more thermostable than that from a mesophile, B. sphaericus, and retained its full activity upon heating at 75 degrees C for at least 60 min and with incubation in pH 5.5-9.5 at 75 degrees C for 10 min. The enzyme can be stored without loss of its activity in a frozen state (-20 degrees C, at pH 7.2) for over 5 months. The optimum pH for the L-alanine deamination and pyruvate amination were around 10.5 and 8.2, respectively. The enzyme exclusively catalyzed the oxidative deamination of L-alanine in the presence of NAD+, but showed low amino acceptor specificity; hydroxypyruvate, oxaloacetate, 2-oxobutyrate and 3-fluoropyruvate are also aminated as well as pyruvate in the presence of NADH and ammonia. Initial velocity and product inhibition studies showed that the reductive amination proceeded through a sequential mechanism containing partially random binding. NADH binds first to the enzyme, and then pyruvate and ammonia bind in a random fashion. The products are sequentially released from the enzyme in the order L-alanine then NAD+. A dead-end inhibition by the formation of an abortive ternary complex which consists of the enzyme, NAD+ and pyruvate was included in the reaction. A possible role of the dead-end inhibition is to prevent the enzyme from functioning in the L-alanine synthesis. The Michaelis constants for the substrates were as follows: NADH, 0.10 mM; pyruvate, 0.50 mM; ammonia, 38.0 mM; L-alanine, 10.5 mM and NAD+, 0.26 mM.