In situ behavior of the pyrimidine pathway enzymes in Saccharomyces cerevisiae. I. Catalytic and regulatory properties of aspartate transcarbamylase

A permeabilization procedure was adapted to allow the in situ determination of aspartate transcarbamylase activity in Saccharomyces cerevisiae. Permeabilization is obtained by treating cell suspensions with small amounts of 10% toluene in absolute ethanol. After washing, the cells can be used directly in the enzyme assays. Kinetic studies of aspartate transcarbamylase (EC 2.1.3.2) in such permeabilized cells showed that apparent Km for substrates and Ki for the feedback inhibitor UTP were only slightly different from those reported using partially purified enzyme. The aspartate saturation curve is hyperbolic both in the presence and absence of UTP. The inhibition by this nucleotide is noncompetitive with respect to aspartate, decreasing both the affinity for this substrate and the maximal velocity of the reaction. The saturation curves for both substrates give parallel double reciprocal plots. The inhibition by the products is linear noncompetitive. Succinate, an aspartate analog, provokes competitive and uncompetitive inhibitions toward aspartate and carbamyl phosphate, respectively. The inhibition by phosphonacetate, a carbamyl phosphate analog, is uncompetitive and noncompetitive toward carbamyl phosphate and aspartate, respectively, but pyrophosphate inhibition is competitive toward carbamyl phosphate and noncompetitive toward aspartate. These results, as well as the effect of the transition state analog N-phosphonacetyl-L-aspartate, all exclude a random mechanism for aspartate transcarbamylase. Most of the data suggest an ordered mechanism except the substrates saturation curves, which are indicative of a ping-pong mechanism. Such a discrepancy might be related to some channeling of carbamyl phosphate between carbamyl phosphate synthetase and aspartate transcarbamylase catalytic sites.     

Reaction mechanism of aspartate transcarbamylase from mouse spleen

The reaction mechanism of aspartate transcarbamylase from mouse spleen has been determined, using steady-state kinetics, isotope-exchange experiments, inhibition studies with a transition-state analog, and product-inhibition studies. Intersecting reciprocal plots obtained when one substrate was varied against different concentrations of the second substrate indicate that the mechanism is sequential. The transition-state analog, N-(phosphonacetyl)-l-aspartate, was a powerful inhibitor of aspartate transcarbamylase, with an inhibition constant (K_i) of 2.6 × 10^?8m at 37 °C and pH 7.4 in 0.05 m Na HEPES buffer. PALA gave competitive inhibition with carbamyl phosphate and noncompetitive inhibition with l-aspartate, indicating that carbamyl phosphate must bind before aspartate for catalysis to occur. A ping-pong mechanism in which carbamyl phosphate binds first was excluded by isotope-exchange experiments, since [³²P]inorganic phosphate was not incorporated into carbamyl phosphate in the absence of aspartate. Product-inhibition studies showed that only inorganic phosphate and carbamyl phosphate gave a competitive pattern; all other combinations of substrate and product gave noncompetitive inhibition patterns when incubations were carried out at subsaturating concentrations of the second substrate. These inhibition patterns showed that carbamyl phosphate binds first, aspartate binds second, carbamyl aspartate dissociates first, and phosphate dissociates second.     

13C isotope effects as a probe of the kinetic mechanism and allosteric properties of Escherichia coli aspartate transcarbamylase.

13C kinetic isotope effects have been measured in carbamyl phosphate for the reaction catalyzed by aspartate transcarbamylase. For the holoenzyme, the value was 1.0217 at zero aspartate, but unity at infinite aspartate, with 4.8 mM aspartate eliminating half of the isotope effect. This pattern proves an ordered kinetic mechanism, with carbamyl phosphate adding before aspartate. The same parameters were observed in the presence of ATP or CTP, showing that there is only one form of active enzyme present, regardless of the presence or absence of allosteric modifiers. These data support the Monod model of allosteric behavior in which the equilibrium between fully active and inactive enzyme is perturbed by selective binding interactions of substrates and modifiers, and there are no enzyme forms having partial activity. Isolated catalytic subunits of the enzyme showed similar 13C isotope effects (1.0240 at zero aspartate, 1.0039 at infinite aspartate, 3.8 mM aspartate causing half of the change from one value to the other), but the finite isotope effect at infinite aspartate shows that the kinetic mechanism is now partly random. With the very slow and poorly bound aspartate analog cysteine sulfinate, the 13C isotope effects were 1.039 for both holoenzyme and catalytic subunits and were not decreased significantly by high levels of cysteine sulfinate. The value of 1.039 is probably close to the intrinsic isotope effect on the chemical reaction, while the kinetic mechanism with this substrate is now fully random because the chemistry is so much slower than release of either reactant from the enzyme.     

Modes of modifier action in E. coli aspartate transcarbamylase

The observed patterns for inhibition by CTP and succinate of equilibrium exchange kinetics with native aspartate transcarbamylase (E. coli) are consistent with an ordered substrate-binding system in which aspartate binds after carbamyl phosphate, and phosphate is released after carbamyl aspartate. ATP selectively stimulates Asp carbamyl-Asp exchange, but not carbamyl phosphate P_i. Initial velocity studies at 5 °, 15 °, and 35 °C were carried out, using modifiers as perturbants of the system. Modifiers alter the Hill n and S_0.5 for aspartate, most markedly at 15 °C but less so at the other temperatures. ATP does increase V under saturating substrate conditions, and substrate inhibition is observed for aspartate. ATP does not make the Hill n = 1 at any temperature. It is proposed that CTP and ATP act by separate mechanisms, not by simply perturbing in opposite directions the equilibrium for aspartate binding. ATP appears to act to increase the rate of aspartate association and dissociation, whereas CTP induces an intramolecular competitive effect in the protein.     

Purification and properties of aspartate transcarbamylase from Mycobacterium smegmatis

Aspartate transcarbamylase (carbamoyl-phosphate: L-aspartate carbamoyltransferase, EC 2.1.3.2) has been purified from Mycobacterium smegmatis TMC 1546 using streptomycin sulphate precipitation, ammonium sulphate precipitation, DE-52 chromatography, second ammonium sulphate precipitation, Sephadex G-200 gel filtration, and aspartate-linked CNBr-activated Sepharose 4B affinity chromatography in successive order. The enzyme was purified 231.6-fold, and the preparation was found to be homogeneous on column chromatography and polyacrylamide gel electrophoresis. The purified enzyme had a molecular weight of 246,000 and was composed of two asymmetrical subunits. The kinetic and regulatory properties of aspartate transcarbamylase from M. smegmatis were also studied. The enzyme was found to be an allosteric in nature with carbamyl phosphate showing positive cooperativity and UMP exhibiting a negative cooperativity. CTP was found to be the most potent inhibitor among nucleotides. Phosphate acted as a non-competitive product inhibitor with respect to aspartate. Succinate and maleate exerted a competitive inhibition when aspartate was the variable substrate.     

Interaction of rose bengal with mung bean aspartate transcarbamylase

The fluorescein dye, rose bengal in the dark: (i) inhibited the activity of mung bean aspartate transcarbamylase (EC 2.1.3.2) in a non-competitive manner, when aspartate was the varied substrate; (ii) induced a lag in the time course of reaction and this hysteresis was abolished upon preincubation with carbamyl phosphate; and (iii) converted the multiple bands observed on polyacrylamide gel electrophoresis of enzyme into a single band. The binding of the dye to the enzyme induced a red shift in the visible spectrum of dye suggesting that it was probably interacting at a hydrophobic region in the enzyme. The dye, in the presence of light, inactivated the enzyme and the inactivation was not dependent on pH. All the effects of the dye could be reversed by UMP, an allosteric inhibitor of the enzyme. The loss of enzyme activity on photoinactivation and the partial protection afforded by N-phosphonoacetyl-L-aspartate, a transition state analog and carbamyl phosphate plus succinate, a competitive inhibitor for aspartate, as well as the reversal of the dye difference spectrum by N-phosphonoacetyl-L-aspartate suggested that in the mung bean aspartate transcarbamylase, unlike in the case ofEscherichia coli enzyme, the active and allosteric sites may be located close to each other.     

Ordered substrate binding and evidence for a thermally induced change in mechanism for E. coli aspartate transcarbamylase

Isotopic exchange kinetics at equilibrium for E. coli native aspartate transcarbamylase at pH 7.8, 30 °C, are consistent with an ordered BiBi substrate binding mechanism. Carbamyl phosphate binds before l-Asp, and carbamyl-aspartate is released before inorganic phosphate. The rate of [¹⁴C]Asp C-Asp exchange is much faster than [³²P]carbamyl phosphate P_i exchange. Phosphate, and perhaps carbamyl phosphate, appears to bind at a separate modifier site and prevent dissociation of active-site bound P_i or carbamyl phosphate. Initial velocity studies in the range of 0–40 °C reveal a biphasic Arrhenius plot for native enzyme: E_a (>15 °C) = 6.3 kcal/ mole and E_a (<15 °C) = 22.1 kcal/mole. Catalytic subunits show a monophasic plot with E_a ? 20.2 kcal/mole. This, with other data, suggests that with native enzyme a conformational change accompanying aspartate association contributes significantly to rate limitation at t > 15 °C, but that catalytic steps become definitively slower below 15 °C. Model kinetics are derived to show that this change in mechanism at low temperature can force an ordered substrate binding system to produce exchange-rate patterns consistent with a random binding system with all exchange rates equal. The nonlinear Arrhenius plot also has important consequences for current theories of catalytic and regulatory mechanisms for this enzyme.     

Studies onplant aspartate transcarbamylase: Regulatory properties of the enzyme from mung bean (Phaseolus aureus) seedlings

Aspartate transcarbamylase (EC 2·1·3·2) purified from mung bean seedlings was used as a model to understand the mechanism of allosteric regulation. The enzyme exhibited homotropic interactions with carbamyl phosphate. Preincubation of the enzyme with aspartate abolished the sigmoidicity of the carbamyl phosphate saturation curve. UMP was the most potent inhibitor of the reaction and was noncompetitive with respect to aspartate. The sigmoidicity of carbamyl phosphate saturation curves increased with increase in UMP concentration. These results were analysed by an iterative least squares procedure. There was no change inV _max values with increase in the UMP concentration, although theK _0·5 values (concentration of carbamyl phosphate required to reach half maximal velocity) increased. This implied that the effect of UMP was on the binding of carbamyl phosphate only and not on the catalytic function of the enzyme. The allosteric properties of the enzyme could be explained in terms ofK system of the symmetry model. The values of the allosteric constantsn, L andc calculated for mung bean enzyme, making use of the Monod equation accounted for all the observed properties. The enzyme appeared to be a tetramer (n=4) and in the absence of ligands was predominantly in theT form (L _o= 2·25). Carbamyl phosphate bound preferentially to theR form (c= 10_?3), while UMP bound preferentially to theT form and hence these two ligands exhibited the typical heterotropic interactions as expected of antagonistic ligands.     

Three residues involved in binding and catalysis in the carbamyl phosphate binding site of Escherichia coli aspartate transcarbamylase

Site-directed mutagenesis was used to create four mutant versions of Escherichia coli aspartate transcarbamylase at three positions in the catalytic chain of the enzyme. The location of all the amino acid substitutions was near the carbamyl phosphate binding site as previously determined by X-ray crystallography. Arg-54, which interacts with both the anhydride oxygen and a phosphate oxygen of carbamyl phosphate, was replaced by alanine. This mutant enzyme was approximately 17,000-fold less active than the wild type, although the binding of substrates and substrate analogues was not altered substantially. Arg-105, which interacts with both the carbonyl oxygen and a phosphate oxygen of carbamyl phosphate, was replaced by alanine. This mutant enzyme exhibited an approximate 1000-fold loss of activity, while the activity of catalytic subunit isolated from this mutant enzyme was reduced by 170-fold compared to the wild-type catalytic subunit. The KD of carbamyl phosphate and the inhibition constants for acetyl phosphate and N-(phosphono-acetyl)-L-aspartate (PALA) were increased substantially by this amino acid substitution. Furthermore, this loss in substrate and substrate analogue binding can be correlated with the large increases in the aspartate and carbamyl phosphate concentrations at half of the maximum observed specific activity, [S]0.5. Gln-137, which interacts with the amino group of carbamyl phosphate, was replaced by both asparagine and alanine. The asparagine mutant exhibited only a small reduction in activity while the alanine mutant was approximately 50-fold less active than the wild type. The catalytic subunits of both these mutant enzymes were substantially more active than the corresponding holoenzymes.(ABSTRACT TRUNCATED AT 250 WORDS)     

A 70-amino acid zinc-binding polypeptide fragment from the regulatory chain of aspartate transcarbamoylase causes marked changes in the kinetic mechanism of the catalytic trimer.

Interaction between a 70-amino acid and zinc-binding polypeptide from the regulatory chain and the catalytic (C) trimer of aspartate transcarbamoylase (ATCase) leads to dramatic changes in enzyme activity and affinity for active site ligands. The hypothesis that the complex between a C trimer and 3 polypeptide fragments (zinc domain) is an analog of R state ATCase has been examined by steady-state kinetics, heavy-atom isotope effects, and isotope trapping experiments. Inhibition by the bisubstrate ligand, N-(phosphonacetyl)-L-aspartate (PALA), or the substrate analog, succinate, at varying concentrations of substrates, aspartate, or carbamoyl phosphate indicated a compulsory ordered kinetic mechanism with carbamoyl phosphate binding prior to aspartate. In contrast, inhibition studies on C trimer were consistent with a preferred order mechanism. Similarly, 13C kinetic isotope effects in carbamoyl phosphate at infinite aspartate indicated a partially random kinetic mechanism for C trimer, whereas results for the complex of C trimer and zinc domain were consistent with a compulsory ordered mechanism of substrate binding. The dependence of isotope effect on aspartate concentration observed for the Zn domain-C trimer complex was similar to that obtained earlier for intact ATCase. Isotope trapping experiments showed that the compulsory ordered mechanism for the complex was attributable to increased "stickiness" of carbamoyl phosphate to the Zn domain-C trimer complex as compared to C trimer alone. The rate of dissociation of carbamoyl phosphate from the Zn domain-C trimer complex was about 10(-2) that from C trimer.(ABSTRACT TRUNCATED AT 250 WORDS)     

A single mutation in the active site swaps the substrate specificity of N-acetyl-L-ornithine transcarbamylase and N-succinyl-L-ornithine transcarbamylase

Transcarbamylases catalyze the transfer of the carbamyl group from carbamyl phosphate (CP) to an amino group of a second substrate such as aspartate, ornithine, or putrescine. Previously, structural determination of a transcarbamylase from Xanthomonas campestris led to the discovery of a novel N-acetylornithine transcarbamylase (AOTCase) that catalyzes the carbamylation of N-acetylornithine. Recently, a novel N-succinylornithine transcarbamylase (SOTCase) from Bacteroides fragilis was identified. Structural comparisons of AOTCase from X. campestris and SOTCase from B. fragilis revealed that residue Glu92 (X. campestris numbering) plays a critical role in distinguishing AOTCase from SOTCase. Enzymatic assays of E92P, E92S, E92V, and E92A mutants of AOTCase demonstrate that each of these mutations converts the AOTCase to an SOTCase. Similarly, the P90E mutation in B. fragilis SOTCase (equivalent to E92 in X. campestris AOTCase) converts the SOTCase to AOTCase. Hence, a single amino acid substitution is sufficient to swap the substrate specificities of AOTCase and SOTCase. X-ray crystal structures of these mutants in complexes with CP and N-acetyl-L-norvaline (an analog of N-acetyl-L-ornithine) or N-succinyl-L-norvaline (an analog of N-succinyl-L-ornithine) substantiate this conversion. In addition to Glu92 (X. campestris numbering), other residues such as Asn185 and Lys30 in AOTCase, which are involved in binding substrates through bridging water molecules, help to define the substrate specificity of AOTCase. These results provide the correct annotation (AOTCase or SOTCase) for a set of the transcarbamylase-like proteins that have been erroneously annotated as ornithine transcarbamylase (OTCase, EC 2.1.3.3).     

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.     

Synthesis and inactivation of carbamyl phosphate synthetase isozymes of Bacillus subtilis during growth and sporulation.

Pyrimidine-repressible carbamyl phosphate synthetase P was synthesized in parallel with aspartate transcarbamylase during growth of Bacillus subtilis on glucose-nutrient broth. Both enzymes were inactivated at the end of exponential growth, but at different rates and by different mechanisms. Unlike the inactivation of aspartate transcarbamylase, the inactivation of carbamyl phosphate synthetase P was not interrupted by deprivation for oxygen or in a tricarboxylic acid cycle mutant. The arginine-repressible isozyme carbamyl phosphate synthetase A was synthesized in parallel with ornithine transcarbamylase during the stationary phase under these growth conditions. Again, both enzymes were subsequently inactivated, but at different rates and by apparently different mechanisms. The inactivation of carbamyl phosphate synthetase A was not affected in a protease-deficient mutatn the inactivation of ornithine transcarbamylase was greatly slowed.     

The overall synthesis of L-5,6-dihydroorotate by multienzymatic protein pyr1-3 from hamster cells. Kinetic studies, substrate channeling, and the effects of inhibitors

In mammals, a trifunctional protein (ME pyr1-32) synthesizes L-5,6-dihydroorotate in three sequential reactions catalyzed by carbamyl phosphate synthetase (EC 2.7.2.9), aspartate transcarbamylase (EC 2.1.3.2), and dihydroorotase (EC 3.5.2.3). 14C-labeled HCO3- has been used as a precursor for the synthesis of L-5,6-dihydroorotate by purified ME pyr1-3, and when this product is converted enzymatically to orotidine 5'-monophosphate, the concentrations of the two intermediates of ME pyr1-3, carbamyl phosphate, and N-carbamyl-L-aspartate, reach steady state concentrations of approximately 0.20 microM and 7.1 microM, respectively. At pH 7.4 in the presence of 0.1 mM 5-phosphoribosyl 1-pyrophosphate and 10% (v/v) glycerol, pure ME pyr1-3 has a Michaelis constant for HCO3- of 0.61 mM and a maximal specific activity of 329 pmol of L-5,6-dihydroorotate synthesized/min/microgram, equivalent to a turnover number of 65.8 mol min-1 (mol of subunit)-1. Consideration of the Km and Vmax values of aspartate transcarbamylase and dihydroorotase determined under the same conditions as the overall rate of synthesis of L-5,6-dihydroorotate by ME pyr1-3, indicates that the local concentrations of carbamyl phosphate at the active site of aspartate transcarbamylase and of N-carbamyl-L-aspartate at the dihydroorotase site must be 2.2-fold and 3.1-fold higher, respectively, than their average concentrations in the bulk solvent. Similar concentrations are predicted by calculation of steady state concentrations from ratios of the rate constants for the three activities. A high local concentration of N-carbamyl-L-aspartate at the third site is also indicated by a 3.6-fold reduction in the transient time for dihydroorotase activity from that predicted. Competition experiments performed with exogenous carbamyl phosphate and N-carbamyl-L-aspartate indicate only partial channeling of these intermediates. The inhibitory effect of N-phosphonacetyl-L-aspartate (PALA), at concentrations up to at least 2.4 microM, upon the aspartate transcarbamylase activity of ME pyr1-3 can be overcome by accumulated carbamyl phosphate. This mechanism for resistance to PALA could be manifest in cells which lack an effective phosphatase activity to hydrolyze carbamyl phosphate. L-Cysteine, a slow acting but potent inhibitor of dihydroorotase in the absence of substrates (Christopherson, R. I., and Jones, M. E. (1980) J. Biol. Chem. 255, 3358-3370), also inactivates dihydroorotate when ME pyr1-3 is synthesizing L-5,6-dihydroorotate.     

Inhibition of biotin carboxylase by a reaction intermediate analog: implications for the kinetic mechanism

The first committed step in long-chain fatty acid synthesis is catalyzed by the multienzyme complex acetyl CoA carboxylase. One component of the acetyl CoA carboxylase complex is biotin carboxylase which catalyzes the ATP-dependent carboxylation of biotin. The Escherichia coli form of biotin carboxylase can be isolated from the other components of the acetyl CoA carboxylase complex such that enzymatic activity is retained. The synthesis of a reaction intermediate analog inhibitor of biotin carboxylase has been described recently (Organic Lett. 1, 99-102, 1999). The inhibitor is formed by coupling phosphonoacetic acid to the 1'-N of biotin. In this paper the characterization of the inhibition of biotin carboxylase by this reaction-intermediate analog is described. The analog showed competitive inhibition versus ATP with a slope inhibition constant of 8 mM. Noncompetitive inhibition was found for the analog versus biotin. Phosphonoacetate exhibited competitive inhibition with respect to ATP and noncompetitive inhibition versus bicarbonate. Biotin was found to be a noncompetitive substrate inhibitor of biotin carboxylase. These data suggested that biotin carboxylase had an ordered addition of substrates with ATP binding first followed by bicarbonate and then biotin.     

Kinetic studies of fructokinase I of pea seeds

Fructokinase I of pea seeds has been purified to homogeneity and the enzyme shown to be monomeric, with a molecular weight of 72,000 +/- 4000. The reaction mechanism was investigated by means of initial velocity studies. Both substrates inhibited the enzyme; the inhibition caused by MgATP was linear-uncompetitive with respect to fructose whereas that caused by D-fructose was hyperbolic-noncompetitive against MgATP. The product D-fructose 6-phosphate caused hyperbolic-noncompetitive inhibition with respect to both substrates. MgADP caused noncompetitive inhibition, which gave intercept and slope replots that were linear with D-fructose but hyperbolic with MgATP. Free Mg2+ caused linear-uncompetitive inhibition when either substrate was varied. L-Sorbose and beta, gamma-methyleneadenosine 5'-triphosphate were used as analogs of D-fructose and MgATP, respectively. Inhibition experiments using these compounds indicated that substrate addition was steady-state ordered, with MgATP adding first. The product inhibition experiments were found to be consistent with a steady-state random release of products. The substrate inhibition caused by MgATP was most likely due to the formation of an enzyme-MgATP-product dead-end complex, whereas that caused by D-fructose was due to alternative pathways in the reaction mechanism. The inhibition caused by Mg2+ can be explained in terms of a dead-end complex with either a central complex or an enzyme-product complex.     

6-phosphofructokinase (pyrophosphate). Properties of the enzyme from Entamoeba histolytica and its reaction mechanism.

The inorganic pyrophosphate-requiring 6-phosphofructokinase of Entamoeba histolytica has been further investigated. The molecular weight of the enzyme is approximately 83,000 and its isoelectric point occurs at pH 5.8 to 6.0. The divalent cation requirement for reaction was explored. In the direction of fructose 6-phosphate formation half-maximal rate required 500 muM magnesium ion; in the direction of fructose bisphosphate formation 8 muM magnesium ion sufficed. ATP, PPi, polyphosphate, acetyl phosphate, or carbamyl phosphate cannot replace PPi as phosphate donor for the conversion of fructose 6-phosphate to fructose bisphosphate. In the direction of fructose 6-phosphate formation arsenate can replace orthophosphate. Isotope exchange studies indicate that little or no exchange occurs between Pi and PPi or between fructose 6-phosphate and fructose bisphosphate in the absence of a third substrate. These findings appear to rule out phosphoenzyme formation and a ping-pong reaction mechanism. PPi, Pi, and fructose bisphosphate are competitive inhibitors of fructose bisphosphate, PPi, and fructose 6-phosphate, respectively. This argues against an ordered mechanism and suggests a random mechanism. Fructose 6-phosphate and Pi were noncompetitive with respect to each other indicating the formation of a dead end complex. These product inhibition relationships are in accord with a Random Bi Bi mechanism.     

Reverse direction substrate kinetics and inhibition studies on the first enzyme of histidine biosynthesis, adenosine triphosphate phosphoribosyltransferase.

Initial velocity steady-state substrate kinetics for ATP phosphoribosyltransferase were determined in the direction reverse to the biosynthetic reaction and are consistent with a sequential kinetic mechanism. Histidine inhibited the reverse reaction cooperatively and completely. Product and alternate product inhibition studies were conducted to elucidate binding order. The alternate product β,γ-methylene ATP was competitive with respect to N¹-phosphoribosyl-ATP and noncompetitive with respect to pyrophosphate. Phosphoribosylpyrophosphate was noncompetitive with respect to both substrates. These data and those of the biosynthetic direction reaction are in satisfactory quantitative agreement with the ordered Bi-Bi kinetic mechanism with ATP or phosphoribosyl-ATP binding to free enzyme.     

Aspartate transcarbamylase de la coquille Saint-Jacques Pecten maximus 1. (Mollusque lamellibranche): Méthode de dosage et variations de l'activité dans le manteau et la gonade

The search of an index for the instantaneous estimate of the in situ growth rate of marine animals led us to attempt to measure the specific activity of aspartate transcarbamylase (ATC). The experiments to test the value of the index were carried out on the scallop Pecten maximus L. p]The first step was to find the optimum conditions for enzyme activity measurement. At 35 °C. the scallop ATC shows an optimum pH of 9 and a K_m of 4.6 × 10^?3 M for aspartate and of 8.0 × 10^?4 M for carbamyl phosphate. The different types of inhibition by the substrates high concentrations and the products suggest an ordered sequential mechanism for the reaction. The decrease in enzyme activity due to metallic ions (Cu²⁺ and Zn²⁺) and to parahydroxymercuribenzoate is compatible with the presence of a sulphydryl group in the active site. p]The variations in ATC levels within the gonad and within the mantle of the scallop were measured and compared with the processes of the sexual maturation and somatic growth in a natural population. For the two tissues, a correlation between the ATC specific activity and the relative growth rate is demonstrated.     

Kinetic mechanism of tRNA nucleotidyltransferase from Escherichia coli.

A kinetic analysis of the incorporation of AMP into tRNA lacking the 3'-terminal residue by tRNA nucleotidyltransferase (EC 2.2.7.25) from Escherichia coli is presented. Initial velocity studies demonstrate that the mechanism is sequential and that high concentrations of tRNA give rise to substrate inhibition which is noncompetitive with respect to ATP. In addition, the substrate inhibition is more pronounced in the presence of pyrophosphate, which suggests the formation of an inhibitory enzyme-pyrophosphate-tRNA complex. Noncompetitive product inhibition is observed between all possible pairs of substrates and products. ADP and alpha,beta-methylene adenosine triphosphate are competitive dead end inhibitors of ATP, while the latter is a noncompetitive dead end inhibitor of the tRNA substrate. A nonrapid equilibrium random mechanism is proposed which is consistent with these data and offers an explanation for the noncompetitive substrate inhibition by tRNA.