Studies on the sucrose phosphate synthetase kinetic mechanism

The kinetic properties of wheat germ sucrose phosphate synthetase, which catalyzes the reaction UDP-glucose + fructose 6-phosphate → UDP + sucrose 6-phosphate 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 was competitive with UDP-glucose and noncompetitive with fructose 6-phosphate. A dead-end inhibitor, inorganic phosphate, was competitive with UDP-glucose and noncompetitive with fructose 6-phosphate. The results of initial velocity and product and dead-end inhibition studies suggested that the addition of substrates to the enzyme follows an ordered mechanism.     

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.     

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.     

Studies on the mechanism of 3-ketosphinganine synthetase.

The biosynthesis of sphinganine and 4-D-hydroxysphinganine was studied in rat liver microsomes and whole cells of yeast (Hansenula ciferri). It was shown in both cases that the condensation of [2,3,3-2H3]serine and palmitic acid yielded long chain bases containing only two deuterium atoms, both of which were located on the terminal (C-1) carbon atom by combined gas-liquid chromatography/mass spectrometry. When the reaction with the liver microsomal system was carried out in 2H2O with the protium species of serine, the sphinganine contained a deuterium atom on C-2. These results suggest that the synthesis of 3-ketosphinganine involves the replacement of the alpha-hydrogen atom and the carboxyl group of serine by a proton from the medium and a palmitoyl group, rather than a previously proposed mechanism in which the alpha-hydrogen of serine is retained. Some stereochemical requirements of 3-ketosphinganine synthetase are discussed.     

Kinetic mechanism of beef pancreatic L-asparagine synthetase

The kinetic mechanism of bovine pancreatic asparagine synthetase was deduced from initial velocity studies and product inhibition studies of both the glutamine-dependent and ammonia-dependent reactions. For the glutamine-dependent pathway, parallel lines were observed in the double reciprocal plot of 1/V vs. 1/[glutamine] at varied aspartate concentrations, and in the plot of 1/V vs. 1/[ATP] at varied aspartate concentrations. Intersecting lines were found for the plot of 1/V vs. 1/[ATP] at varied glutamine concentrations. Product inhibition patterns, including dual inhibitor studies for measuring the synergistic effects of multiproduct inhibition, were used to support an ordered bi-uni-uni-ter ping-pong mechanism. Glutamine and ATP sequentially bind, followed by the release of glutamate and the addition of aspartate. Pyrophosphate, AMP, and asparagine are then sequentially released. When the ammonia-dependent reaction was studied, it was found that the mechanism was significantly different. NH3 bound first followed by a random addition of ATP and aspartate. Pyrophosphate, AMP, and asparagine were then sequentially released as in the glutamine-utilizing mechanism. From these studies, a comprehensive mechanism has been proposed through which either glutamine or NH3 can provide nitrogen for asparagine production from aspartate.     

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 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 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.     

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.     

Sucrose metabolism in green algae. I. The presence of sucrose synthetase and sucrose phosphate synthetase.

The presence of sucrose synthetase and sucrose phosphate synthetase has been demonstrated in two species of green algae: Chlorella vulgaris and Scenedesmus obliquus. Partial purification from crude extracts allowed the determination of the kinetic constants of algae enzymes. They are very similar to the ones reported for enzymes from higher plants.     

Kinetic studies on human cysteinyl-tRNA synthetase.

The detailed pH and temperature kinetics of human term placenta cysteinyl-tRNA synthetase (EC 6.1.1.16) were studied. The ATP-PPi exchange reaction catalyzed by the cysteinyl-tRNA synthetase was highly dependent on temperature, pH, and ionic strength. The Arrhenius plot at temperatures between 5 degrees and 40 degrees was linear, giving an activation energy of 19 +/- 2.5 Kcal/mol. The pH dependence of the kinetic parameters Km and Vmax was investigated. Apparent pKa value of 6.4 was observed in the pH-dependence of Vmax/Km plot. The pH versus Vmax plot showed two apparent pKa values of about 5.8 and 7.8. Van't Hoff's enthalpies were used to differentiate the nature of the possible groups responsible for the ionization. These results are valuable for the selection of chemical modifying reagents in characterizing the amino acid residues involved in substrate (nucleotide) binding or catalysis.     

Sucrose metabolism in green algae I. The presence of sucrose synthetase and sucrose phosphate synthetase

Summary The presence of sucrose synthetase and sucrose phosphate synthetase has been demonstrated in two species of green algae:Chlorella vulgaris andScenedesmus obliquus. Partial purification from crude extracts allowed the determination of the kinetic constants of algae enzymes. They are very similar to the ones reported for enzymes from higher plants.Dedicated toLuis F. Leloir on his seventieth birthday.     

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.     

Examination of the mechanism of sucrose synthetase by positional isotope exchange

The mechanism of the sucrose synthetase reaction has been probed by the technique of positional isotope exchange. [beta-18O2, alpha beta-18O]UDP-Glc has been synthesized starting from oxygen-18-labeled phosphate and the combined activities of carbamate kinase, hexokinase, phosphoglucomutase, and uridine diphosphoglucose pyrophosphorylase. The oxygen-18 at the alpha beta-bridge position of the labeled UDP-Glc has been shown to cause a 0.014 ppm upfield chemical shift in the 31P NMR spectrum of both the alpha- and beta-phosphorus atoms in UDP-Glc relative to the unlabeled compound. The chemical shift induced by each of the beta-nonbridge oxygen-18 atoms was 0.030 ppm. Incubation of [beta-18O2, alpha beta-18O]UDP-Glc with sucrose synthetase in the presence and absence of 2,5-anhydromannitol did not result in any significant exchange of an oxygen-18 from the beta-nonbridge position to the anomeric oxygen of the glucose moiety. It can thus be concluded that either sucrose synthetase does not catalyze the cleavage of the scissile carbon-oxygen bond of UDP-Glc in the absence of fructose or, alternatively, the beta-phosphoryl group of the newly formed UDP is rotationally immobilized.