Studies of medium-chain fatty acyl-coenzyme A synthetase. Enzyme fraction II: mechanism of reaction and specific properties

1. The mechanism of reaction of fatty acyl-CoA synthesis catalysed by fatty acyl-CoA synthetase from ox liver (fraction II; Bar-Tana, Rose & Shapiro, 1968) was investigated by a kinetic study of CoA disappearance dependent on butyrate plus ATP or butyryl-AMP (overall and partial reaction b respectively). 2. Contrary to findings with another enzyme (fraction I), a Bi Uni Uni Bi Ping Pong mechanism (Cleland, 1963a,b,c) corresponding to Berg's (1956) scheme of reaction was eliminated and an ordered Ter Ter mechanism with an A-C-B (standing for ATP, CoA and butyrate respectively) sequence of substrate entry for the overall reaction was established for fraction II. Partial reaction (b) was found to follow the ;Iso-Theorell-Chance' mechanism. 3. Also, in contrast with results obtained with fraction I, no allosteric properties could be demonstrated with fraction II.     

Assay, purification, properties and mechanism of action of γ-glutamylcysteine synthetase from the liver of the rat and Xenopus laevis

1. An improved radioassay for glutathione synthetase and gamma-glutamylcysteine synthetase was developed. 2. Xenopus laevis liver gamma-glutamylcysteine synthetase was purified 324-fold by saline-bicarbonate extraction, protamine sulphate precipitation, CM-cellulose and DEAE-cellulose column chromatography, and gel filtration. 3. Rat liver gamma-glutamylcysteine synthetase was purified 11400-fold by a procedure similar to that employed for the Xenopus laevis enzyme. 4. Rat liver gamma-glutamylcysteine synthetase activity was inhibited by GSH and activated by glycine. These effects, which were not found in the enzyme from Xenopus laevis, may have a regulatory significance. 5. Isotope-exchange experiments revealed fundamental differences in the partial reactions catalysed by the rat and Xenopus laevis synthetases. The enzyme from Xenopus laevis appears to follow a Bi Bi Uni Uni Ping Pong mechanism, with glutamyl-enzyme as intermediate before the addition of cysteine and the release of gamma-glutamylcysteine. The results for the rat liver enzyme are consistent with a Tri Tri sequential mechanism.     

Steady-state and pre-steady-state kinetic analysis of Mycobacterium tuberculosis pantothenate synthetase.

Pantothenate synthetase (EC 6.3.2.1), encoded by the panC gene, catalyzes the essential ATP-dependent condensation of D-pantoate and beta-alanine to form pantothenate in bacteria, yeast and plants. Pantothenate synthetase from Mycobacterium tuberculosis was expressed in E. coli, purified to homogeneity, and found to be a homodimer with a subunit molecular mass of 33 kDa. Initial velocity, product, and dead-end inhibition studies showed the kinetic mechanism of pantothenate synthetase to be Bi Uni Uni Bi Ping Pong, with ATP binding followed by D-pantoate binding, release of PP(i), binding of beta-alanine, followed by the release of pantothenate and AMP. Michaelis constants were 0.13, 0.8, and 2.6 mM for D-pantoate, beta-alanine, and ATP, respectively, and the turnover number, k(cat), was 3.4 s(-1). The formation of pantoyl adenylate, suggested as a key intermediate by the kinetic mechanism, was confirmed by (31)P NMR spectroscopy of [(18)O]AMP produced from (18)O transfer using [carboxyl-(18)O]pantoate. Single-turnover reactions for the formation of pyrophosphate and pantothenate were determined using rapid quench techniques, and indicated that the two half-reactions occurred with maximum rates of 1.3 +/- 0.3 and 2.6 +/- 0.3 s(-)(1), respectively, consistent with pantoyl adenylate being a kinetically competent intermediate in the pantothenate synthetase reaction. These data also suggest that both half-reactions are partially rate-limiting. Reverse isotope exchange of [(14)C]-beta-alanine into pantothenate in the presence of AMP was observed, indicating the reversible formation of the pantoyl adenylate intermediate from products.     

Kinetic studies of bovine liver fructose-1,6-bisphosphatase.

Initial rate kinetic studies with bovine liver fructose-1,6-bisphosphatase were carried out in both directions of the reaction to determine the sequence of product release from the enzyme. Product inhibition by fructose-6-P was found to be S-linear, I-linear noncompetitive relative to fructose-1,6-bisphosphate, whereas inorganic orthophosphate was determined to be linear competitive with respect to the substrate. The kinetics of the reverse reaction were studied by coupling the phosphatase reaction to the aldolase, triosephosphate isomerase, and glycerolphosphate dehydrogenase reactions. The kinetic results were found to be in harmony with the Uni Bi ordered and random sequential mechanisms as well as a Uni Bi ping-pong mechanism. The nomenclature is that of Cleland (Cleland, W.W. (1963) Biochim. Biophys. Acta 67, 104-137). However, nonkinetic considerations, when taken together with the kinetic results, suggest that the steady state ordered Uni Bi mechanism is the most likely possibility. There is evidence that isomerization of the binary complex of enzyme and phosphate occurs in the kinetic mechanism. Although magnesium is required for the reverse reaction, there is no evidence to suggest that the enzyme discriminates between the magnesium-associated or divalent cation-free forms of the substrates.     

Studies on medium-chain fatty acyl-coenzyme A synthetase. Enzyme fraction I: mechanism of reaction and allosteric properties

1. The mechanism of butyrate activation catalysed by an enzyme fraction derived from ox liver particles (fraction I; Bar-Tana, Rose & Shapiro, 1968) was studied by an analysis of the initial-velocity pattern of the overall reaction and found to conform to the Bi Uni Uni Bi Ping Pong model (Cleland, 1963a,b,c) in agreement with the reaction scheme proposed by Berg (1956). 2. A homotropic co-operative effect was exerted by CoA on fraction I, whereas ATP and AMP functioned as heterotropic co-operative ligands with respect to butyryl-AMP-dependent CoA disappearance. On the other hand, PP(i) and butyryl-CoA showed antagonistic heterotropic effects when tested under similar conditions. With respect to the overall reaction CoA and ATP could be shown to function as co-operative homotropic modifiers. 3. Two interchangeable conformational states of the enzyme are therefore presumed to exist, state R, having a higher affinity for CoA and ATP and thus preferentially catalysing butyryl-AMP-dependent CoA disappearance (partial reaction b), and state T, favoured by the presence of PP(i), catalysing the formation of ATP from butyryl-AMP and PP(i) (partial reaction a) with greater efficiency. 4. These findings serve to explain the opposite effects of ATP on the partial reactions, as well as the inhibition by CoA and ATP of ATP formation (reaction a) and by PP(i) of the butyryl-AMP-dependent CoA disappearance (reaction b) (Bar-Tana et al. 1968). 5. The possible analogy of these observations to amino acid-activating and other similar systems is discussed.     

Kinetic mechanism of arginyl-tRNA synthetase from human placenta

Like arginyl-tRNA synthetases from other organisms, human placental arginyl-tRNA synthetase catalyzes the arginine-dependent ATP-PPi exchange reaction only in the presence of tRNA. We have investigated the order of substrate addition and product release of this human enzyme in the tRNA aminoacylation reaction by using initial velocity experiments and dead-end product inhibition studies. The kinetic patterns obtained are consistent with a random Ter Ter sequential mechanism, instead of the common Bi Uni Uni Bi ping-pong mechanism for all other human aminoacyl-tRNA synthetases so far investigated in this respect.     

Characterization of the propionyl-CoA synthetase (PrpE) enzyme of Salmonella enterica: residue Lys592 is required for propionyl-AMP synthesis

The propionyl-CoA synthetase (PrpE) enzyme of Salmonella enterica catalyzes the first step of propionate catabolism, i.e., the activation of propionate to propionyl-CoA. The PrpE enzyme was purified, and its kinetic properties were determined. Evidence is presented that the conversion of propionate to propionyl-CoA proceeds via a propionyl-AMP intermediate. Kinetic experiments demonstrated that propionate was the preferred acyl substrate (kcat/Km = 1644 mM(-1) x s(-1)). Adenosine 5'-propyl phosphate was a potent inhibitor of the enzyme, and inhibition kinetics identified a Bi Uni Uni Bi Ping Pong mechanism for the reaction catalyzed by the PrpE enzyme. Site-directed mutagenesis was used to change the primary sequence of the wild-type protein at positions G245A, P247A, K248A, K248E, G249A, K592A, and K592E. Mutant PrpE proteins were purified, and the effects of the mutations on enzyme activity were investigated. Both PrpEK592 mutant proteins (K592A and K592E) failed to convert propionate to propionyl-CoA, and plasmids containing these alleles of prpE failed to restore growth on propionate of S. enterica carrying null prpE alleles on their chromosome. Both PrpEK592 mutant proteins converted propionyl-AMP to propionyl-CoA, suggesting residue K592 played no discernible role in thioester bond formation. To the best of our knowledge, these mutant proteins are the first acyl-CoA synthetases reported that are defective in adenylation activity.     

Properties and reaction mechanism of C4 leaf pyruvate,Pi dikinase

The properties and reaction mechanism of maize leaf pyruvate,Pi dikinase are described. Km values were determined for the forward reaction substrates, pyruvate, ATP, and Pi, at pH 7.4 and 8.0 and for reverse reaction substrates at pH 7.4. Enzyme activity was almost totally dependent on added monovalent cations in both directions. NH+4 was most effective, with Ka values of about 0.38 mM for the forward reaction and 2 mM for the reverse reaction. K+ also completely activated the enzyme in the forward direction (Ka = 8 mM) but only partially activated in the reverse direction. Na+ had little effect on either reaction. The pH optimum for the forward reaction was about 8.2; the reverse reaction optimum was about 6.9. Maximum activity for the reverse direction was about twice the maximum forward direction rate. From data on the requirements for the ATP-AMP exchange reaction, on the mechanism of inhibition of the forward reaction by PEP, AMP, and PPi, and from the kinetics of the interaction of varying certain substrate pairs, it was concluded that the maize leaf pyruvate,Pi dikinase reaction proceeded by the two-step Bi Bi Uni Uni mechanism. This differs from the mechanism of catalysis by the bacterial enzyme.     

Differences between microsomal and mitochondrial-matrix palmitoyl-coenzyme A hydrolase, and palmitoyl-L-carnitine hydrolase from rat liver.

Palmitoyl-CoA hydrolase (EC 3.1.2.2) and palmitoyl-L-carnitine hydrolase (EC 3.1.1.28) activities from rat liver were investigated. 1. Microsomal and mitochondrial-matrix palmitoyl-CoA hydrolase activities had similar pH and temperature optima, although the activities showed different temperature stability. They were inhibited by Pb2+ and Zn2+. The palmitoyl-CoA hydrolase activities in microsomal fraction and mitochondrial matrix were differently affected by the addition of Mg2+, Ca2+, Co2+, K+ and Na+ to the reaction mixture. ATP, ADP and NAD+ stimulated the microsomal activity and inhibited the mitochondrial-matrix enzyme. The activity of both the microsomal and mitochondrial-matrix hydrolase enzymes was specific for long-chain fatty acyl-CoA esters (C12-C18), with the highest activity for palmitoyl-CoA. The apparent Km for palmitoyl-CoA was 47 microM for the microsomal enzyme and 17 microM for the mitochondrial-matrix enzyme. 2. The palmitoyl-CoA hydrolase and palmitoyl-L-carnitine hydrolase activities of microsomal fraction had similar pH optima and were stimulated by dithiothreitol, but were affected differently by the addition of Pb2+, Mg2+, Ca2+, Mn2+ and cysteine. The two enzymes had different temperature-sensitivities. 3. The data strongly suggest that palmitoyl-CoA hydrolase and palmitoyl-L-carnitine hydrolase are separate microsomal enzymes, and that the hydrolysis of palmitoyl-CoA in the microsomal fraction and mitochondria matrix was catalysed by two different enzymes.     

Sheep kidney pyruvate carboxylase. Studies on the coupling of adenosine triphosphate hydrolysis and CO2 fixation.

Initial velocity and isotope exchange studies confirmed that the over-all reaction, like that catalyzed by pyruvate carboxylase purified from rat liver and chicken liver, was a nonclassical Ping Pong Bi Bi Uni Uni sequence with ATP and HCO3-binding randomly in the Bi Bi partial reaction. Three possible mechanisms for the coupling of ATP hydrolysis and CO2 fixation are considered: (i) Mechanism i, a concerted mechanism without the formation of a kinetically significant or detectable intermediate; (ii) Mechanism ii, activation of the enzyme by ATP to form an activated phosphoenzyme complex which can react with HCO3- by formation of a phosphorylated intermediate. On the basis of other evidence, an activated intermediate containing the ADP moiety was considered improbable. Evidence is presented which indicates that an isotopic exchange between ATP and ADP in the absence of added orthophosphate is not a property of the sheep kidney enzyme. This observation removed the need to postulate either that this exchange is an abortive reaction, or that there is a compulsory formation of a phosphoenzyme intermediate. Two analogues of ADP, alpha,beta-methylene adenosine diphosphate, and adenosine 5'-phosphosulfate, have been used to provide further evidence against Mechanism ii. Both compounds were competitive inhibitors with respect to MgATP2- (Ki values respectively, 0.58 mM and 3.0 mM, compared with 0.17 mM for ADP), but neither could be phosphorylated by the enzyme. Neither analogue could replace ADP in the HCO3-: oxalacetate isotopic exchange reaction, indicating that phosphorylation of ADP is necessary for this exchange to occur, and that Mechanism ii is not applicable. Since Mechanism iii involves formation of a carbonly phosphate intermediate, analogues of this compound, namely, carbamyl phosphate and phosphonacetic acid were used to examine this pathway. The fact that the enzyme catalyzed the synthesis of ATP from ADP and carbamyl phosphate, and that phosphonacetic acid was a noncompetitive inhibitor with respect to MgATP2- (Ki = 0.5 mM) provides strong evidence that a carbonyl phosphate derivative is involved in the reaction mechanism. However, we have not found from initial velocity studies evidence for the formation of this intermediate, and it may therefore have only a transient existence in an essentially concerted reaction.     

Influence of supramolecular structure on the enzyme mechanisms of rat liver lysyl-tRNA synthetase-catalyzed reactions. Synthesis of P1,P4-bis(5'-adenosyl)tetraphosphate

Lysyl-tRNA synthetase, dissociated from the multienzyme complexes of aminoacyl-tRNA synthetases from rat liver, was previously found to be 6-fold more active than the synthetase complex in the enzymatic synthesis of P1,P4-bis(5'-adenosyl)tetraphosphate. The bi-substrate and product inhibition kinetics of the reaction are analyzed. Free lysyl-tRNA synthetase exhibits distinctly different kinetic patterns from those of an 18 S synthetase complex containing lysyl-tRNA synthetase. The 18 S synthetase complex shows kinetic patterns which are consistent with an ordered Bi Uni Uni Bi ping-pong mechanism. Free lysyl-tRNA synthetase shows kinetic patterns consistent with a random mechanism. The differences in the enzymatic properties are attributed to the organization of the supramolecular structure of the synthetase complex. The results suggest that association of the synthetases may affect the mechanisms of the synthesis of AppppA.     

Kinetic studies on the enzyme (S)-hydroxynitrile lyase from hevea brasiliensis using initial rate methods and progress curve analysis

(S)-Hydroxynitrile lyase (EC 4.1.2.39) from Hevea brasiliensis(rubber tree) catalyzes the reversible cleavage of cyanohydrins to aldehydes or ketones and prussic acid (HCN). Enzyme kinetics in both directions was studied on a model system with mandelonitrile, benzaldehyde, and HCN using two different methods-initial rate measurements and progress curve analysis. To discriminate between possible mechanisms with the initial rate method, product inhibition was studied. Benzaldehyde acts as a linear competitive inhibitor against mandelonitrile whereas HCN shows S-linear I-parabolic mixed-type inhibition. These results indicate an Ordered Uni Bi mechanism with the formation of a dead-end complex of enzyme, (S)-mandelonitrile and HCN. Prussic acid is the first product released from the enzyme followed by benzaldehyde. For progress curve analysis, a kinetic model of an Ordered Uni Bi mechanism including a dead-end complex, enzyme inactivation, and the chemical parallel reaction was set up, which described the experimental values very well. From the reaction rates obtained the kinetic constants were calculated and compared with the ones obtained from the initial rate method. Good agreement could be achieved between the two methods supporting the suggested mechanism. Copyright 1999 John Wiley & Sons, Inc.     

Steady-state investigations of the mechanism of histidinol dehydrogenase.

Initial velocities of the histidinol dehydrogenase reaction (EC 1.1.1.23) were measured as a function of the concentrations of the substrates histidinol and NAD+ and in the presence and absence of the product NADH. The data are consistent with a Bi Uni Uni Bi Ping Pong mechanism. The kinetic constants of this mechanism were determined; Km for histidinol was found to be 14 microM and for NAD+ 0.7 mV; Ki for NAD+ was 0.4 mM.     

Partial purification and characterization of fatty acid binding protein(s) in Escherichia coli membranes and reconstitution of fatty acid transport system

Fatty acid binding protein(s) in E. coli membranes was solubilized and partially purified by oleate-AH Sepharose 4B column chromatography. The binding of palmitate to the protein was saturable. The protein bound all fatty acids with chain lengths of 10-18 tested, and its maximum activity was observed with palmitate. The incorporation of the protein into liposomes which contained a system for acyl-CoA synthesis significantly increased the uptake of the extracellular [14C] palmitate by the liposomes in comparison with the liposomes without the protein. The uptake of [14C] palmitate was also saturable, and the accumulated radioactive compound was found to be palmitoyl-CoA.     

Reaction pathway of bovine aortic lysyl oxidase

The catalysis of amine oxidation by lysyl oxidase has been probed to assess for the likely order of substrate binding and product release and to discriminate between mechanistic alternatives previously proposed for other copper-dependent amine oxidases using molecular oxygen as a substrate. Lineweaver-Burk plots revealed a pattern of parallel lines when the oxidation of n-butylamine was followed at different fixed concentrations of oxygen consistent with a "ping-pong" kinetic mechanism in which the aldehyde is produced and released before the binding of oxygen, the second substrate. Initial burst experiments revealed the ability of lysyl oxidase to form and release n-butyraldehyde in amounts stoichiometric with functional active site content in the absence of oxygen, consistent with the ping-pong kinetics obtained. Reciprocal plots of n-butylamine oxidation in the presence of fixed concentrations of the reaction products were consistent with a Uni Uni Uni Bi ping-pong kinetic mechanism with the aldehyde being the first, H2O2 the second, and ammonia the last departing product. Moreover, spectral studies of the oxidation of p-hydroxybenzylamine by lysyl oxidase indicated that the enzyme does not process the amine substrate to a noncovalently bound p-hydroxybenzaldimine intermediate subsequently to be hydrolyzed to p-hydroxybenzaldehyde. The kinetic mechanism of lysyl oxidase thus appears to be similar to those described for diamine oxidase and pig plasma monoamine oxidase.     

Mechanism of thermal adaptation of membrane lipids in Tetrahymena pyriformis NT-1. Possible evidence for temperature-mediated induction of palmitoyl-CoA desaturase.

The regulatory mechanism of a key enzyme, palmitoyl-CoA desaturase, involved in the adaptation to temperature shift was investigated by labeling Tetrahymena pyriformis cells with [14C]palmitic acid. The rate of conversion of [14C]palmitate to [14C]palmitoleate was shown to be dependent on incubation temperature and also to be maximal at 2 h after the shift 39.5 to 15 degrees C. Addition of cycloheximide before the temperature shift produced no increase in desaturation of [14C]palmitate after the shift. These data would provide evidence for temperature-triggered increase of palmitoyl-CoA desaturase level and are also discussed in relation to membrane fluidity.     

Kinetic model for synthesis of fructosyl-stevioside using suspended β-fructofuranosidase

The synthesis experiments of fructosyl-stevioside were conducted under the various conditions of the initial concentrations of the substrates and the enzyme. The transfructosylation of stevioside with sucrose and the hydrolyses of sucrose and fructosyl-stevioside simultaneously occurred. The fructosyl-stevioside synthesis was inhibited by the side products, glucose and fructose. A kinetic model was constructed by considering the Ping-Pong Bi Bi mechanism for the transfructosylation, the apparent Ordered Uni Bi mechanism for the hydrolysis and the competitive inhibition by the side products. The model constants were estimated by fitting the model equations with the experimental results for the sucrose hydrolysis and the fructosyl-stevioside synthesis. The model can predict not only the appropriate conditions to efficiently synthesize the fructosyl-stevioside, but also the reaction time giving the maximum conversion.     

A product-inhibition study of the mechanism of mitochondrial octanoyl-coenzyme A synthetase

By a study of the product-inhibition kinetics of the octanoyl-CoA synthetase from ox liver mitochondria, evidence was obtained consistent with the hypothesis that the enzyme reacts by a Bi Uni Uni Bi Ping Pong type of mechanism in which the order of addition and evolution of substrates and products is CoA, octanoate, octanoyl-CoA, ATP, PP_i and AMP. There is also evidence that more than one molecule of CoA can add to the enzyme and that it may act as an allosteric activator.     

Glyceraldehyde-3-phosphate dehydrogenase (NADP) from Sinapis alba: Steady state kinetics

Substrate interaction and product inhibition kinetics of the forward reaction of glyceraldehyde-3-phosphate dehydrogenase (NADP) (EC 1.2.1.13) from Sinapis alba suggest an Uni Uni Uni Bi Ping Pong mechanism (NAD(P)H on, glyceraldehyde-3-phosphate off, 1,3-diphosphoglycerate on, phosphate off, NAD(P)⁺ off) with an apparent Theorell Chance displacement between 1,3-diphosphoglycerate and phosphate. The proposed mechanism predicts the existence of stable enzyme-NAD(P)⁺ and acyl-enzyme complexes as obligatory intermediates. A comparison of the present findings on the NADP-enzyme with an earlier kinetic analysis of the NAD-specific enzyme from plants (EC 1.2.1.12) by other authors shows that the kinetic mechanisms for the two enzymes, although similar in principle (both show Ping Pong kinetics), differ in some details.     

Effect of monovalent anions on the mechanism of phenol hydroxylase

The mechanism of phenol hydroxylase (EC 1.14.13.7) has been studied by steady state and rapid reaction kinetic techniques. Both techniques give results consistent with the Bi Uni Uni Bi ping-pong mechanism proposed for other flavin-containing aromatic hydroxylases. The enzyme binds phenolic substrate and NADPH in that order, followed by reduction of the flavin and release of NADP+. A transient charge transfer complex between reduced enzyme and NADP+ can be detected. Molecular oxygen then reacts with the reduced enzyme-substrate complex. Two to three flavin-oxygen intermediates can be detected in the oxidative half-reaction depending on the substrate, provided monovalent anions are present. Oxygen transfer is complete with the formation of the second intermediate. Based on its UV absorption spectrum and on the fact that oxygen transfer has taken place, the last of these intermediates is presumably the flavin C(4a)-hydroxide. Monovalent anions are uncompetitive inhibitors of phenol hydroxylase. The mechanistic step most affected is the dehydration of the flavin C(4a)-hydroxide to give oxidized enzyme. Chloride also kinetically stabilizes the blue flavin semiquinone of phenol hydroxylase during photoreduction. These data suggest binding of monovalent anions results in stabilization of a proton on the N(5) position of the flavin.