pH-dependence of the triose phosphate isomerase reaction

1. Some kinetic properties of aspartate transcarbamoylase (EC 2.1.3.2), that had been purified approx. 20-fold from wheat germ, were studied. 2. A plot of enzyme activity against pH showed a low maximum at pH8.4 and a second, higher, maximum at pH10.5. A plot of percentage inhibition by 0.2mm-UMP against pH was approximately parallel to the plot of activity against pH, except that between pH6.5 and 7.5 the enzyme was insensitive to 0.2mm-UMP. 3. Kinetics were studied in detail at pH10.0 and 25 degrees C. In the absence of UMP, initial-rate plots were hyperbolic when the concentration of either substrate was varied. UMP decreased both V(max.) and K(m) in plots of initial rate against l-aspartate concentration, but the plots remained hyperbolic. However, UMP converted plots of initial rate against carbamoyl phosphate concentration into a sigmoidal shape, without significantly affecting V(max.). Plots of initial rate against UMP concentration were also sigmoidal. 4. The theoretical model proposed by Monod et al. (1965) gave a partial explanation of these results. When quasi-equilibrium conditions were assumed analysis in terms of this model suggested a trimeric enzyme binding the allosteric ligands, carbamoyl phosphate and UMP, nearly exclusively to the R and T conformational states respectively, and existing predominantly in the R state when ligands were absent. However, the values of the Hill coefficients for the co-operativity of each allosteric ligand were somewhat less than those predicted by the theory. 5. Some of the implications of these results are discussed, and the enzyme is contrasted with the well-known aspartate transcarbamoylase of Escherichia coli.     

Ionization of amino acid residues involved in the catalytic mechanism of aspartate transcarbamoylase.

The chemical and kinetic mechanisms of the reaction catalyzed by the catalytic trimer of aspartate transcarbamoylase have been examined. The variation of the kinetic parameters with pH indicated that at least four ionizing amino acid residues are involved in substrate binding and catalysis. The pH dependence of K(ia) for carbamoyl phosphate and the K(i) for N-(phosphonoacetyl)-L- aspartate revealed that a protonated residue with a pK value of 9.0 is required for the binding of carbamoyl phosphate. However, the variation with pH of K(i) for succinate, a competitive inhibitor of aspartate, and for cysteine sulfinate, a slow substrate, showed that a single residue with a pK value of 7.3 must be protonated for binding these analogues and, by inference, aspartate. The profile of log V against pH displayed a decrease in reaction rate at low and high pH, suggesting that two groups associated with the Michaelis complex, a deprotonated residue with a pK value of 7.2 and a protonated group with a pK value of 9.5, are involved in catalysis. By contrast, the catalytically productive form of the enzyme-carbamoyl phosphate complex, as illustrated in the bell-shaped pH dependence of log (V/K)(asp), is one in which a residue with a pK value of 7.0 must be protonated while a group with a pK value of 9.1 is deprotonated. This interpretation is supported by the results from the temperature dependence of the V and V/K profiles and from the pH dependence of pK(i) for the aspartate analogues.(ABSTRACT TRUNCATED AT 250 WORDS)     

Steady-state kinetics and isotope effects on the mutant catalytic trimer of aspartate transcarbamoylase containing the replacement of histidine 134 by alanine.

A detailed kinetic analysis of the catalytic trimer of aspartate transcarbamoylase containing the active site substitution H134A was performed to investigate the role of His 134 in the catalytic mechanism. Replacement of histidine by alanine resulted in decreases in the affinities for the two substrates, carbamoyl phosphate and aspartate, and the inhibitor succinate, by factors of 50, 10, and 6, respectively, and yielded a maximum velocity that was 5% that of the wild-type enzyme. However, the pK values determined from the pH dependence of the kinetic parameters, log V and log (V/K) for aspartate, the pK(i) for succinate, and the pK(ia) for carbamoyl phosphate, were similar for both the mutant and the wild-type enzymes, indicating that the protonated form of His 134 does not participate in binding and catalysis between pH 6.2 and 9.2. 13C and 15N isotope effects were studied to determine which steps in the catalytic mechanism were altered by the amino acid substitutions. The 13(V/K) for carbamoyl phosphate exhibited by the catalytic trimer containing alanine at position 134 revealed an isotope effect of 4.1%, probably equal to the intrinsic value and, together with quantitative analysis of the 15N isotope effects, showed that formation of the tetrahedral intermediate is rate-determining for the mutant enzyme. Thus, His 134 plays a role in the chemistry of the reaction in addition to substrate binding. The initial velocity pattern for the reaction catalyzed by the H134A mutant intersected to the left of the vertical axis, negating an equilibrium ordered kinetic mechanism.(ABSTRACT TRUNCATED AT 250 WORDS)     

Pyrimidine nucleotide biosynthesis in Phaseolus aureus. Enzymic aspects of the control of carbamoyl phosphate synthesis and utilization

1. Aspartate transcarbamoylase from 4-day-old radicles of Phaseolus aureus was purified 190-fold by (NH(4))(2)SO(4) fractionation, DEAE-cellulose and DEAE-Sephadex chromatography and Sephadex-gel filtration. The partially purified enzyme, which required P(i) for maximum stability, had an apparent molecular weight of 83000+/-5000. 2. Uridine nucleotides were found to inhibit the activity; UMP was the most potent inhibitor, followed by UDP and UTP. No other nucleotide was found to affect the enzyme, nor could UMP inhibition be overcome by adding another nucleotide. Aspartate gives a hyperbolic substrate-saturation curve, both with and without UMP. The nucleotide inhibitor is non-competitive with respect to this substrate. Carbamoyl phosphate also yields a hyperbolic substrate-saturation curve in the absence of feedback inhibitor, but when UMP is added a sigmoidal pattern results, and the inhibition is competitive with carbamoyl phosphate. 3. The degree of inhibition by UMP is not affected by p-chloromercuribenzoate, urea, mild heat pretreatment or change in pH over the range 8.5-10.5, but is affected by temperature. 4. The aspartate analogue, succinate, both activates and inhibits the reaction, depending on the concentrations of aspartate and succinate used. 5. Kinetic studies with the partially purified enzyme showed that the K(m) for carbamoyl phosphate (0.091 mm) is much lower than that for aspartate (1.7mm). A sequential reaction mechanism was inferred from product-inhibition kinetics, with carbamoyl phosphate binding to the enzyme before aspartate, and the product, carbamoylaspartate, being released ahead of P(i). Initial-velocity studies gave a set of parallel reciprocal plots, compatible with an essentially irreversible step occurring before the binding of aspartate.     

Factors accelerating pyrimidine production in <Emphasis Type="Italic">Deinococcus radiophilus</Emphasis>

In studying the pyrimidine synthesising pathway in Deinococcus radiophilus two instances of anomalous behaviour were observed. One was the strikingly different results obtained for two types of assay for carbamoyl phosphate synthetase. Both depend on the fixation of ¹⁴C from the substrate bicarbonate to give radioactive products. In the coupled assay the carbamoyl phosphate product of the enzyme is converted to carbamoyl aspartate in the presence of aspartate and aspartate transcarbamoylase. In the direct assay aspartate is omitted from the reaction mixture and the carbamoyl phosphate is converted to urea. It was found that the radioactive counts in the direct assay were about 5% of those measured in the coupled assay. The second anomaly was that omission of glutamine from both assay mixtures had no significant effect on the fixation of radioactive carbon. These results suggested that aspartate amino-N could be the source of nitrogen for glutamine synthesis by a substrate-channelled pathway which delivered glutamine to carbamoyl phosphate synthetase, and that externally added glutamine could not access its binding site on the enzyme.     

Wheat-germ aspartate transcarbamoylase. The effect of ligands on the inactivation of the enzyme by trypsin and denaturing agents

In the absence of added ligands aspartate transcarbamoylase (EC 2.1.3.2) from wheat germ is inactivated fairly rapidly by trypsin, by heat (60 degrees C), by highly alkaline conditions (pH11.3) and by sodium dodecyl sulphate. Addition of UMP alone, at low concentrations, decreases the rate of inactivation by each of these agents significantly. Carbamoyl phosphate alone does not alter the rate of inactivation by trypsin and by the detergent, but it antagonizes the effect of UMP in protecting the enzyme against these agents. These results have been interpreted to mean that two conformational states are reversibly accessible to the enzyme, namely an easily inactivated state favoured in the presence of carbamoyl phosphate and a more resistant state favoured in the presence of UMP. In the absence of ligands the enzyme is in the easily inactivated conformation. At very high concentrations l-aspartate also protects the enzyme but to a smaller extent than UMP. Some implications of these results are discussed.     

A radioisotopic method for the assay of aspartate carbamoyltransferase and carbamoyl phosphate

The use of [¹⁴C]aspartate of high specific activity and thin-layer chromatography on polyethyleneimine cellulose for the separation of carbamoyl aspartate from aspartate has enabled the measurement of aspartate carbamoyltransferase and carbamoyl phosphate synthase activities and carbamoyl phosphate concentrations in extracts from Escherichia coli. The assay method described is sensitive to the formation of about 1 pmol of carbamoyl aspartate.     

T-state inhibitors of E. coli aspartate transcarbamoylase that prevent the allosteric transition

Escherichia coli aspartate transcarbamoylase (ATCase) catalyzes the committed step in pyrimidine nucleotide biosynthesis, the reaction between carbamoyl phosphate (CP) and l-aspartate to form N-carbamoyl-l-aspartate and inorganic phosphate. The enzyme exhibits homotropic cooperativity and is allosterically regulated. Upon binding l-aspartate in the presence of a saturating concentration of CP, the enzyme is converted from the low-activity low-affinity T state to the high-activity high-affinity R state. The potent inhibitor N-phosphonacetyl-l-aspartate (PALA), which combines the binding features of Asp and CP into one molecule, has been shown to induce the allosteric transition to the R state. In the presence of only CP, the enzyme is the T structure with the active site primed for the binding of aspartate. In a structure of the enzyme-CP complex (T(CP)), two CP molecules were observed in the active site approximately 7A apart, one with high occupancy and one with low occupancy. The high occupancy site corresponds to the position for CP observed in the structure of the enzyme with CP and the aspartate analogue succinate bound. The position of the second CP is in a unique site and does not overlap with the aspartate binding site. As a means to generate a new class of inhibitors for ATCase, the domain-open T state of the enzyme was targeted. We designed, synthesized, and characterized three inhibitors that were composed of two phosphonacetamide groups linked together. These two phosphonacetamide groups mimic the positions of the two CP molecules in the T(CP) structure. X-ray crystal structures of ATCase-inhibitor complexes revealed that each of these inhibitors bind to the T state of the enzyme and occupy the active site area. As opposed to the binding of Asp in the presence of CP or PALA, these inhibitors are unable to initiate the global T to R conformational change. Although the best of these T-state inhibitors only has a K(i) value in the micromolar range, the structural information with respect to their mode of binding provides important information for the design of second generation inhibitors that will have even higher affinity for the active site of the T state of the enzyme.     

The contribution of threonine 55 to catalysis in aspartate transcarbamoylase.

Heavy-atom isotope effects and steady-state kinetic parameters were measured for the catalytic trimer of an active site mutant of aspartate transcarbamoylase, T55A, to assess the role of Thr 55 in catalysis. The binding of carbamoyl phosphate to the T55A mutant was decreased by 2 orders of magnitude relative to the wild-type enzyme whereas the affinities for aspartate and succinate were not markedly altered. This indicates that Thr 55 plays a significant role in the binding of CbmP. If, as had been suggested previously, Thr 55 assists in the polarization of the carbonyl group of CbmP, the carbon isotope effect for the T55A mutant should increase relative to that observed for the wild-type enzyme. However, the opposite is seen, indicating that Thr 55 is not involved in stabilizing the oxyanion in the transition state. Quantitative analysis of a series of 13C and 15N isotope effects suggested that the rate-determining step in the reaction catalyzed by T55A trimer may be a conformational change in the protein subsequent to formation of the Michaelis complex. Thus, Thr 55 may facilitate a conformational change in the enzyme that is a prerequisite for catalysis. An altered active site environment in the binary and Michaelis complexes with T55A trimer is reflected in the pH profiles for log V, log (V/K)asp, and pK(i) succinate, show a displacement in the pK values of ionizing residues involved in aspartate binding and catalysis relative to the wild-type enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)     

An ultraviolet spectrophotometric assay for aspartate transcarbamylase

A procedure for determining the activity of aspartate transcarbamylase, based upon the greater ultraviolet absorbancy of the products of the reaction catalyzed compared to the reactants, was devised. Extinction coefficients were determined at 205, 210, and 215 nm for the compounds carbamoyl aspartate, acetyl aspartate, and aspartate. These values formed the quantitative basis for a spectrophotometric assay in which an enzymatic reaction is monitored at one of these wavelengths. Use of this procedure was illustrated in four kinetic experiments with the allosteric aspartate transcarbamylase from Escherichia coli, and the nonallosteric catalytic subunit of this enzyme: aspartate saturation curve, arsenate saturation curve (reverse reaction), allosteric activation by a transition-state analog employing acetyl phosphate as substrate, and carbamoyl phosphate progress curve (substrate depletion in the presence of excess cosubstrate). Owing to changes in absorbance on the order of 1000 liter mol^?1 cm^?1 concomitant with the reaction, the sensitivity of the method is comparable to that of many procedures already in the literature.     

Immobilization by Polyurethane of Pseudomonas dacunhae Cells Containing l-Aspartate beta-Decarboxylase Activity and Application to l-Alanine Production

Whole cells of Pseudomonas dacunhae containing l-aspartate beta-decarboxylase activity were immobilized by mixing a cell suspension with a liquid isocyanate-capped polyurethane prepolymer (Hypol; W. R. Grace & Co., Lexington, Mass.). The immobilized cell preparation was used to convert l-aspartic acid to l-alanine. Properties of the immobilized P. dacunhae cells containing aspartate beta-decarboxylase activity were investigated with batch reactors. Retention of enzyme activity was observed to be as much as 100% when cell lysis was allowed to occur before immobilization. The pH and temperature optima were determined to be 5.5 and 45 degrees C, respectively. Immobilized P. dacunhael-aspartate beta-decarboxylase activity was stabilized by the addition of 0.1 mM pyridoxal-5-phosphate and 0.1 mM alpha-ketoglutaric acid to a 1.7 M ammonium aspartate (pH 5.5) substrate solution. Under conditions of semicontinuous use in a batch reactor, a 2.5% loss in immobilized l-aspartate beta-decarboxylase activity was observed over a 31-day period.     

The role of an active site histidine in the catalytic mechanism of aspartate transcarbamoylase

Although the allosteric enzyme aspartate transcarbamoylase from Escherichia coli has been the focus of numerous physical and enzymological studies for over 2 decades, the catalytic mechanism is still poorly understood. There has been much speculation regarding the role of a conserved histidine residue at position 134 which recent crystallographic studies have implicated in the catalytic mechanism as a general acid or as a general base. We have used a combination of site-directed mutagenesis, 13C-isotope incorporation, and high field NMR to probe the role of His134 in the catalytic mechanism of aspartate transcarbamoylase. By comparing the wild-type catalytic trimer with that from a partially active mutant in which His134 is replaced by alanine, we have assigned the 13C resonance for His134 in the wild-type enzyme. This residue is shown to have a pK less than 6, indicating that the imidazole ring is unprotonated at pH values optimal for enzymatic activity (pH 8.0). This result eliminates the possibility of His134 participating as a general acid in the carbamoyl transfer mechanism. Since the crystallographic studies indicate that His134 is close enough to hydrogen-bond to the carbonyl of the liganded bisubstrate analog N-(phosphonacetyl)-L-aspartate, the imidazole ring would be oriented as to make it unlikely that the N1 lone pair of electrons could participate in general base catalysis. Moreover, if His134 is implicated in base catalysis, we would have expected a much greater loss of activity upon its replacement by alanine. Perhaps the role of His134 is merely to help position the carbonyl group of carbamoyl phosphate for nucleophilic attack by the alpha-amino group of aspartate.     

Function of threonine-55 in the carbamoyl phosphate binding site of Escherichia coli aspartate transcarbamoylase

Carbamoyl phosphate is held in the active site of Escherichia coli aspartate transcarbamoylase by a variety of interactions with specific side chains of the enzyme. In particular, the carbonyl group of carbamoyl phosphate interacts with Thr-55, Arg-105, and His-134. Site-specific mutagenesis was used to create a mutant version of the enzyme in which Thr-55 was replaced by alanine in order to help define the role of this residue in the catalytic mechanism. The Thr-55----Ala holoenzyme exhibits a 4.7-fold reduction in maximal observed specific activity, no alteration in aspartate cooperativity, and a small reduction in carbamoyl phosphate cooperativity. The mutation also causes 14-fold and 35-fold increases in the carbamoyl phosphate and aspartate concentrations required for half the maximal observed specific activity, respectively. Circular dichroism spectroscopy has shown that saturating carbamoyl phosphate does not induce a conformational change in the Thr-55----Ala holoenzyme as it does for the wild-type holoenzyme. The kinetic properties of the Thr-55----Ala catalytic subunit are altered to a greater extent than the mutant holoenzyme. The mutant catalytic subunit cannot be saturated by either substrate under the experimental conditions. Furthermore, as opposed to the wild-type catalytic subunit, the Thr-55----Ala catalytic subunit shows cooperativity for aspartate and can be activated by N-(phosphonoacetyl)-L-aspartate in the presence of low concentrations of aspartate and high concentrations of carbamoyl phosphate. As deduced by circular dichroism spectroscopy, the conformation of the Thr-55----Ala catalytic subunit in the absence of active-site ligands is distinctly different from the wild-type catalytic subunit.(ABSTRACT TRUNCATED AT 250 WORDS)     

Partial purification and properties of a highly specific trehalose phosphate phosphatase from Mycobacterium smegmatis

A specific trehalose phosphate phosphatase was purified approximately 50-fold from Mycobacterium smegmatis. The enzyme had a pH optimum of about 7.0 and was stimulated by Mg(2+). The optimum concentration of Mg(2+) was about 1.5 x 10(-3)m. Of other divalent cations tested, only Co(2+) showed some activity. The K(m) for trehalose phosphate was found to be about 1.5 x 10(-3)m. The enzyme showed slight activity toward mannose-6-P and fructose-6-P but was inactive on a large number of other phosphorylated compounds. Citrate was a competitive inhibitor of the enzyme both with respect to trehalose phosphate concentration and Mg(2+) concentration. This inhibition appears to be due to chelation of Mg(2+) by this compound. Ethylenediaminetetraacetic acid and NaF were also inhibitors of the enzyme, but these inhibitions were noncompetitive.     

New assay for enzymatic phosphate release: application to aspartate transcarbamylase and other enzymes

The colorimetric method for phosphate determination described in the preceding paper is adapted for the assay of orthophosphate liberated in the aspartate transcarbamylase reaction. The method provides for simple, accurate, and sensitive measurement of enzyme activity. The assay uses ammonium molybdate and zinc acetate to form a colored complex with the enzymatically released phosphate; mild conditions which minimize the nonenzymatic background degradation of the substrate, carbamoyl phosphate, are used. Since the assay procedure is relatively rapid, it is especially attractive in situations where results are desired immediately. The method can be used for the assay of any enzyme which releases inorganic phosphate, even in the presence of labile organophosphate compounds.     

Studies on alkaline phosphatase: Inhibition by phosphate derivatives and the substrate specificity

1. The kinetics of inhibition of calf-intestinal alkaline phosphatase by inorganic phosphate, fluorophosphate, inorganic pyrophosphate, beta-glycerophosphate and adenosine 5'-triphosphate in the range pH8-10 were investigated. The reference substrate was 4-methylumbelliferyl phosphate. 2. The inhibitions were ;mixed' in that both K(m) and V were affected, but the competitive element was by far the stronger. 3. In each case the pH profile for the competitive K(i) was similar to the pH profile for K(m). Since the K(m) and K(i) values both change 100-fold over the pH range 8-10, it is concluded that the inhibitors compete with the substrate for the same active site. 4. It was also found that the enzyme preparation hydrolysed fluorophosphate, pyrophosphate and adenosine 5'-triphosphate as readily as it hydrolysed 4-methylumbelliferyl phosphate and beta-glycerophosphate. Each pH-activity curve, however, had a different shape, but with the exception of pyrophosphate the activity approached the same maximum value at high pH. 5. Attempts to separate the phosphomonoesterase and pyrophosphatase activities by column chromatography were not successful, and the results of other experiments listed suggest that the two activities are a property of the same enzyme. 6. The effect of Mg(2+) ions is briefly mentioned: the phosphomonoesterase activity is enhanced whereas the pyrophosphatase and adenosine triphosphatase activities are strongly inhibited in the presence of excess of Mg(2+) ions.     

Development of a continuous assay and steady-state characterization of Escherichia coli threonine synthase

Threonine synthase (TS) catalyzes the hydrolysis of O-phospho-L-homoserine (OPHS) to produce L-threonine (L-Thr) and inorganic phosphate. Here, we report a simplified purification protocol for the OPHS substrate and a continuous, coupled-coupled, spectrophotometric TS assay. The sequential actions of threonine deaminase (TD) and hydroxyisocaproate dehydrogenase (HO-HxoDH) convert the L-Thr product of TS to α-ketobutyrate (α-KB) and then to 2-hydroxybutyrate, respectively, and are monitored as the decrease in absorbance at 340 nm resulting from the concomitant oxidation of β-nicotinamide adenine dinucleotide (NADH) to NAD(+) by HO-HxoDH. The effect of pH on the activities of Escherichia coli TD and Lactobacillus delbrueckii HO-HxoDH was determined to establish this continuous assay as suitable for steady-state characterization and to facilitate the optimization of coupling enzyme concentrations under different assay conditions to enable studies of TS across phyla. To validate this assay, TS from E. coli was characterized. The kinetic parameters (k(cat)=4s(-1) and K(m)=0.34 mM) and the pH optimum of 8.7, determined using the continuous assay, are consistent with values reported for this enzyme based on the discontinuous malachite green assay. The k(cat)/K(m)(OPHS) versus pH profile of E. coli TS is bell-shaped, and the apparent pK(a) values for the acidic and basic limbs are 7.1 and 10.4, respectively.     

Cloning, expression and characterisation of Erwinia carotovora L-asparaginase

Bacterial L-asparaginases (E.C. 3.5.1.1) have been used as therapeutic agents in the treatment of acute childhood lymphoblastic leukaemia. L-asparaginase from Erwinia carotovora NCYC 1526 (ErA) was cloned and expressed in E. coli. The enzyme was purified to homogeneity by a two-step procedure comprising cation-exchange chromatography and affinity chromatography on immobilised L-asparagine. The enzymatic properties of the recombinant enzyme were investigated and the kinetic parameters (K(m), k(cat)) for a number of substrates were determined. Molecular modelling studies were also employed to create a model of ErA, based on the known structure of the Erwinia chrysanthemi enzyme. The molecular model was used to help interpret biochemical data concerning substrate specificity and catalytic mechanism of the enzyme. The kinetic parameters of selected substrates were determined at various pH values, and the pH-dependence profiles of V(max) and V(max)/K(m) were analyzed. The pH-dependence of V(max) shows one transition in the acidic pH range with pK(a)=5.4, and the pH-dependence of V(max)/K(m) exhibits two transitions with pK(a)=5.4 and 8.5. Based on analysis of alternative substrates and molecular modelling studies, it was concluded that the pK(a) at the acidic pH range corresponds to the active site residues Asp115 or Glu82, whereas the pK(a) observed at the alkaline pH range is not due to substrate amino group ionisation, but rather is the result of enzyme ionisation. The effect of temperature and viscosity on the catalytic activity of the enzyme was also investigated and it was concluded that the rate-limiting step of the catalytic reaction is relevant to structural transitions of the protein. Thermodynamic analysis of the activity data showed that the activation energies are dependent on the substrate, and entropy changes appear to be the main determinant contributing to substrate specificity.     

Function of serine-52 and serine-80 in the catalytic mechanism of Escherichia coli aspartate transcarbamoylase

Carbamoyl phosphate is held in the active site of Escherichia coli aspartate transcarbamoylase by a variety of interactions with specific side chains of the enzyme. In particular, oxygens of the phosphate of carbamoyl phosphate interact with Ser-52, Thr-53 (backbone), Arg-54, Thr-55, and Arg-105 from one catalytic chain, as well as Ser-80 and Lys-84 from an adjacent chain in the same catalytic subunit. In order to define the role of Ser-52 and Ser-80 in the catalytic mechanism, two mutant versions of the enzyme were created with Ser-52 or Ser-80 replaced by alanine. The Ser-52----Ala holoenzyme exhibits a 670-fold reduction in maximal observed specific activity, and a loss of both aspartate and carbamoyl phosphate cooperativity. This mutation also causes 23-fold and 5.6-fold increases in the carbamoyl phosphate and aspartate concentrations required for half the maximal observed specific activity, respectively. Circular dichroism spectroscopy indicates that saturating carbamoyl phosphate does not induce the same conformational change in the Ser-52----Ala holoenzyme as it does for the wild-type holoenzyme. The kinetic properties of the Ser-52----Ala catalytic subunit are altered to a lesser extent than the mutant holoenzyme. The maximal observed specific activity is reduced by 89-fold, and the carbamoyl phosphate concentration at half the maximal observed velocity increases by 53-fold while the aspartate concentration at half the maximal observed velocity increases 6-fold.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.