Kinetic mechanism of argininosuccinate synthetase

The kinetic mechanism of bovine liver argininosuccinate synthetase has been determined at pH 7.5. The initial velocity and product and dead-end inhibition patterns are consistent with the ordered addition of MgATP, citrulline, and aspartate, followed by the ordered release of argininosuccinate, MgPPi, and AMP. The mechanism is also in accord with the formation of citrulline-adenylate as a reactive intermediate [O. Rochovansky, and S. Ratner, (1967) J. Biol. Chem. 242, 3839-3849]. No evidence was obtained for nonlinear double-reciprocal plots with any of the three substrates.     

Purification and characterization of beef pancreatic asparagine synthetase

Bovine pancreatic asparagine synthetase has been partially purified using ammonium sulfate fractionation, DEAE ion-exchange, Cibacron Blue affinity chromatography, and HPLC anion-exchange chromatography to a specific activity of 170 nmol asparagine produced min-1 mg protein-1, or 1400-fold, from a crude homogenate. Using HPLC size exclusion chromatography, an apparent molecular weight of 110,000-120,000 was determined. An aspartyl-adenylate intermediate was found to occur by demonstrating an 18O transfer from [18O]Asp to AMP that was detected with 31P NMR. A number of divalent metals were found to be able to replace magnesium with retention of activity, but none produced as high an activity as Mg2+, and the stoichiometry of the ATP/Mg2+ ratio was found to be 1. The chloride ion was found to stimulate the glutamine-dependent and glutaminase reactions, but the ammonia-dependent reaction was inhibited. Chloride appeared to be a competitive inhibitor with respect to ammonia and produced negative cooperativity.     

Studies on sucrose synthetase. Kinetic mechanism

The kinetic properties of Helianthus tuberosus sucrose synthetase, which catalyzes the reaction UDP-glucose + fructose = UDP + sucrose, have been studied. A plot of the reciprocal initial velocity versus reciprocal substrate concentration gave a series of intersecting lines indicating a sequential mechanism. Product inhibition studies showed that UDP-glucose was competitive with UDP, whereas fructose was competitive with sucrose and uncompetitive with UDP. On the other hand, a dead-end inhibitor, salicine, was competitive with sucrose and uncompetitive with UDP. The results of initial velocity, product, and dead-end inhibition studies suggested that the addition of substrates to the enzyme follows an ordered mechanism.     

Studies of the ammonia-dependent reaction of beef pancreatic asparagine synthetase

We have studied the asparagine synthetase reaction with regard to the ammonia-dependent production of asparagine. Hydroxylamine was shown to be an alternate substrate for the asparagine synthetase reaction, and some of its kinetic properties were examined. The ammonia-dependent reaction was examined with regard to inhibition by asparagine. It was found that asparagine inhibition was partial competitive with respect to ammonia, regardless of the concentration of aspartate. However, when MgATP was not saturating, the inhibition by asparagine became linear competitive. These results were interpreted to be consistent with a kinetic mechanism for asparagine synthetase where ammonia is bound to the enzyme followed by MgATP causing asparagine release.     

Kinetic mechanism of glutathione synthetase from Arabidopsis thaliana

Glutathione synthetase (GS) catalyzes the ATP-dependent formation of the ubiquitous peptide glutathione from gamma-glutamylcysteine and glycine. The bacterial and eukaryotic GS form two distinct families lacking amino acid sequence homology. Moreover, the detailed kinetic mechanism of the bacterial and the eukaryotic GS remains unclear. Here we have overexpressed Arabidopsis thaliana GS (AtGS) in an Escherichia coli expression system and purified the recombinant enzyme for biochemical characterization. AtGS is functional as a homodimeric protein with steady-state kinetic properties similar to those of other eukaryotic GS. The kinetic mechanism of AtGS was investigated using initial velocity methods and product inhibition studies. The best fit of the observed data was to the equation for a random Ter-reactant mechanism in which dependencies between the binding of some substrate pairs were preferred. The binding of either ATP or gamma-glutamylcysteine increased the binding affinity of AtGS for the other substrate by 10-fold. Likewise, the binding of ATP or glycine increased binding affinity for the other ligand by 3.5-fold. In contrast, binding of either glycine or gamma-glutamylcysteine causes a 6.7-fold decrease in binding affinity for the second molecule. Product inhibition studies suggest that ADP is the last product released from the enzyme. Overall, these observations are consistent with a random Ter-reactant mechanism for the eukaryotic GS in which the binding order of certain substrates is kinetically preferred for catalysis.     

Kinetic mechanism of glutaminyl-tRNA synthetase from human placenta

The order of interaction of substrates and products with human placental glutaminyl-tRNA synthetase was investigated in the aminoacylation reaction by using the steady-state kinetic methods. The initial velocity patterns obtained from both the glutamine-ATP and glutamine-tRNA substrate pairs were intersecting, whereas ATP and tRNA showed double competitive substrate inhibition. Dead-end inhibition studies with an ATP analog, tripolyphosphate, showed uncompetitive inhibition when tRNA was the variable substrate. The product inhibition studies revealed that PPi was an uncompetitive inhibitor with respect to tRNA. The noncompetitive inhibition by AMP versus tRNA was converted to uncompetitive by increasing the concentration of glutamine from 0.05 to 0.5 mM. These and other kinetic patterns obtained from the present study, together with our earlier finding that this human enzyme catalyzed the ATP-PPi exchange reaction in the absence of tRNA, enable us to propose a unique two-step, partially ordered sequential mechanism, with tRNA as the leading substrate, followed by random addition of ATP and glutamine. The products may be released in the following order: AMP, PPi and then glutaminyl-tRNA. The proposed mechanism involves both a quarternary complex including all three substrates and the intermediary formation of an enzyme-bound aminoacyl adenylate, common to the usual sequential and ping-pong mechanisms, respectively, for other aminoacyl-tRNA synthetases.     

Kinetic mechanism of Escherichia coli carbamoyl-phosphate synthetase.

The kinetic mechanism of Escherichia coli carbamoyl-phosphate synthetase has been determined at pH 7.5, 25 degrees C. With ammonia as the nitrogen source, the initial velocity and product inhibition patterns are consistent with the ordered addition of MgATP, HCO3-, and NH3. Phosphate is then released and the second MgATP adds to the enzyme, which is followed by the ordered release of MgADP, carbamoyl phosphate, and MgADP. With glutamine as the ammonia donor, the patterns are consistent with a two-site mechanism in which glutamine binds randomly to the small molecular weight subunit producing glutamate and ammonia. Glutamate is released and the ammonia is transferred to the larger subunit. Carbamoyl-phosphate synthetase has also been shown to require a free divalent cation for full activity.     

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.     

Kinetic mechanism of asparagine synthetase from Vibrio cholerae

Asparagine synthetase B (AsnB) catalyzes the formation of asparagine in an ATP-dependent reaction using glutamine or ammonia as a nitrogen source. To obtain a better understanding of the catalytic mechanism of this enzyme, we report the cloning, expression, and kinetic analysis of the glutamine- and ammonia-dependent activities of AsnB from Vibrio cholerae. Initial velocity, product inhibition, and dead-end inhibition studies were utilized in the construction of a model for the kinetic mechanism of the ammonia- and glutamine-dependent activities. The reaction sequence begins with the ordered addition of ATP and aspartate. Pyrophosphate is released, followed by the addition of ammonia and the release of asparagine and AMP. Glutamine is simultaneously hydrolyzed at a second site and the ammonia intermediate diffuses through an interdomain protein tunnel from the site of production to the site of utilization. The data were also consistent with the dead-end binding of asparagine to the glutamine binding site and PP(i) with free enzyme. The rate of hydrolysis of glutamine is largely independent of the activation of aspartate and thus the reaction rates at the two active sites are essentially uncoupled from one another.     

Multiple forms of rat liver L-asparagine synthetase.

Addition of NaF or MFP to rat hepatocytes resulted in a decrease in lactate and in an increase in glucose, 3 and 2-phosphoglycerate production. When dihydroxyacetone was present in the incubation medium both NaF and MFP increased the production of glucose, fructose-1,6-diphosphate, 3 and 2 phosphoglycerate, with a decrease in pyruvate and lactate. In the presence of lactate, glucose production increased only in the presence of MFP, but there was a 8–10 fold increase in the level of phosphoenol pyruvate with both NaF and MFP. The crossover data indicated that the activity of some of the glycolytic enzymes may be inhibited in the presence of NaF and MFP.     

Kinetic mechanism of the beta-lactam synthetase of Streptomyces clavuligerus

Streptomyces clavuligerus beta-lactam synthetase (beta-LS) was recently demonstrated to catalyze an early step in clavulanic acid biosynthesis, the ATP/Mg(2+)-dependent intramolecular closure of the beta-amino acid N(2)-(carboxyethyl)-L-arginine (CEA) to the monocyclic beta-lactam deoxyguanidinoproclavaminic acid (DGPC). Here we investigate the steady-state kinetic mechanism of the beta-LS-catalyzed reaction to better understand this unprecedented secondary metabolic enzyme. Initial velocity patterns were consistent with a sequential ordered bi-ter kinetic mechanism. Product inhibition studies with PP(i) and DGPC demonstrated competitive inhibition versus their cognate substrates ATP and CEA, respectively, and noncompetitive inhibition against their noncognate substrates. To clarify the order of substrate binding, the truncated substrate analogue N(2)-(carboxymethyl)-L-arginine was synthesized and demonstrated uncompetitive inhibition versus ATP and competitive patterns versus CEA. These data are consistent with ordered substrate binding, with ATP binding first, an abortive enzyme-DGPC complex, and PP(i) released as the last product. The pH dependence of V and V/K was determined and suggests that residues with a pK of 6.5 and 9.3 must be ionized for optimal activity. These observations were considered in the context of investigations of the homologous primary metabolic enzyme asparagine synthetase B, and a chemical mechanism is proposed that is consistent with the kinetic mechanism.     

Inhibition of L-asparagine synthetase by mucochloric and mucobromic acids.

Mucochloric and mucobromic acids are powerful inhibitors of tumoral and pancreatic L-asparagine synthetases. Two nitrogen donors, L-glutamine and ammonia, can be used by these enzymes; at a concentration of 1 mmol/l, mucochloric and mucobromic acids preferentially inhibit the utilization of ammonia as opposed to L-glutamine in vitro. Using the tumoral enzyme, kinetic analysis revealed that mucochloric acid produced inhibition which was apparently noncompetitive with ammonia but competitive with L-glutamine. In molar excess, L-glutamine and dithiothreitol effectively antagonized such inhibition; dialysis, however, failed to reverse established inhibition. These findings, suggest that the drugs operate by covalent attachment to crucial sulfhydryl functions on the enzyme.     

Kinetic mechanism of beef heart ubiquinol:cytochrome c oxidoreductase.

The electron transfer from ubiquinol-2 to ferricytochrome c mediated by ubiquinol:cytochrome c oxidoreductase [E.C. 1.10.2.2] purified from beef heart mitochondria, which contained one equivalent of ubiquinone-10 (Q10), was investigated under initial steady-state conditions. The Q10-depleted enzyme was as active as the Q10-containing one. Double reciprocal plots for the initial steady-state rate versus one of the two substrates at various fixed levels of the other substrate gave parallel straight lines in the absence of any product. Intersecting straight lines were obtained in the presence of a constant level of one of the products, ferrocytochrome c. The other product, ubiquinone-2, did not show any significant effect on the enzymic reaction. Ferrocytochrome c non-competitively inhibited the enzymic reaction against either ubiquinol-2 or ferricytochrome c. These results indicate a Hexa-Uni ping-pong mechanism with one ubiquinol-2 and two ferricytochrome c molecules as the substrates, which involves the irreversible release of ubiquinone-2 as the first product and the irreversible isomerization between the release of the first ferrocytochrome c and the binding of the second ferricytochrome c. Considering the cyclic electron transfer reaction mechanism, this scheme suggests that the binding of quinone or quinol to the enzyme and electron transfer between the iron-sulfur center and cytochrome c1 are rigorously controlled by the electron distribution within the enzyme.     

Kinetic evidence for half-of-the-sites reactivity in tRNATrp aminoacylation by tryptophanyl-tRNA synthetase from beef pancreas

The aminoacylation reaction catalyzed by the dimeric tryptophanyl-tRNA synthetase from beef pancreas was studied under pre-steady-state conditions by the quenched-flow method. The transfer of tryptophan to tRNATrp was monitored by using preformed enzyme-bis(tryptophanyl adenylate) complex. Combinations of either unlabeled or L-[14C]tryptophan-labeled tryptophanyl adenylate and of aminoacylation incubation mixtures containing either unlabeled tryptophan or L-[14C]tryptophan were used. We measured either the formation of a single labeled aminoacyl-tRNATrp per enzyme subunit or the turnover of labeled aminoacyl-tRNATrp synthesis. Four models were proposed to analyze the experimental data: (A) two independent and nonequivalent subunits; (B) a single active subunit (subunits presenting absolute "half-of-the-sites reactivity"); (C) alternate functioning of the subunits (flip-flop mechanism); (D) random functioning of the subunits with half-of-the-sites reactivity. The equations corresponding to the formation of labeled tryptophanyl-tRNATrp under each labeling condition were derived for each model. By use of least-squares criteria, the experimental curves were fitted with the four models, and it was possible to disregard models B and C as likely mechanisms. Complementary experiments, in which there was no significant excess of ATP-Mg over the enzyme-adenylate complex, emphasized an activator effect of free L-tryptophan on the rate of aminoacylation. This result disfavored model A. Model D was in agreement with all data. The analyses showed that the transfer step was not the major limiting reaction in the overall aminoacylation process.     

An investigation of the mechanism of activation of tryptophan by tryptophanyl-tRNA synthetase from beef pancreas

Exposure of dark-grown Euglena to white or red light, but not blue light, produced a twofold increase in the specific activity of citrate synthase. A 400-fold purification of mitochondrial citrate synthase (subunit Mr = 44000) was achieved from cells of Euglena gracilis by affinity chromatography on ATP-activated agarose. Antisera, raised against the homogeneously pure enzyme, were used to demonstrate that the increase in citrate synthase activity on exposure of dark-grown cells to light resulted from an increase in citrate synthase protein. Anti-(citrate synthase) was used to detect precursor citrate synthase resulting from the translation of total polyadenylated RNA from Euglena in a cell-free rabbit reticulocyte lysate system. Citrate synthase mRNA was found to be present in cells at all stages of regreening. However, extraction and translation of polyadenylated RNA from free polysomes isolated from darkgrown and regreening cells demonstrated that appreciable translation of citrate synthase mRNA was only occurring in regreening cells.