Aspartate transcarbamoylase: loss of homotropic but not heterotropic interactions upon modification of the catalytic subunit with a bifunctional reagent.

The role of conformational changes in the allosteric mechanism of aspartate transcarbamoylase from Escherichia coli was studied by reacting the isolated catalytic subunit with the bifunctional reagent tartryl diazide. Two derivatives differing moderately in substrate affinity were obtained depending on whether the reaction was conducted in the presence or absence of the substrate analogue succinate and carbamoyl phosphate. The modification was not accompanied by aggregation or dissociation. The modified catalytic subunits retained the ability to reassociate with unmodified regulatory subunits and produced hybrids similar in size to the native enzyme. These hybrids were appreciably sensitive to the allosteric effectors ATP and CTP but unlike native enzyme showed no cooperativity in substrate binding. The Michaelis constants of these hybrids for aspartate were intermediate between that of the isolated catalytic subunit and that of the relaxed state. Activation by ATP was caused by a reduction in Km to the value characteristic of the relaxed state whereas CTP inhibited by lowering the Vmax. The properties of the hybrids are strikingly similar to the modified enzyme obtained by Kerbiriou and Hervé from cells grown in the presence of 2-thiouracil. However, the crucial modifications are found in the regulatory subunits of the enzyme studied by these authors whereas they are located in the catalytic subunits of the hybrids reported here. Our results suggest that interactions between the catalytic and regulatory subunits have considerable effects on the state of the substrate binding sites in the native enzyme.     

An aspartate transcarbamylase lacking catalytic subunit interactions. Study of conformational changes by ultraviolet absorbance and circular dichroism spectroscopy.

A modified form of aspartate transcarbamylase is synthesized by Escherichia coli in the presence of 2-thiouracil which does not exhibit homotropic cooperative interactions between active sites yet retains heterotropic cooperative interactions due to nucleotide binding. The conformational changes induced in the modified enzyme by the binding of different ligands (substrates, substrate analogs, a transition state analog, and nucleotide effectors) were studied using ultraviolet absorbance and circular dichroism difference spectroscopy. Comparison of the results for the modified enzyme and its isolated subunits to those for the native enzyme and its isolated subunits showed that the conformational changes detected by these methods are qualitatively similar in the two enzymes. Comparison of the absorbance difference spectra due to the binding of a transition substrate analog to the intact native or modified enzymes to the corresponding results for the isolated subunits suggested that ligand binding causes an increased exposure to solvent of certain tyrosyl and phenylalanyl residues in the intact enzymes but not in the isolated subunits. This result is consistent with a diminution of subunit contacts due to substrate binding in the course of homotropic interactions in the native enzyme. Such conformational changes, though perhaps necessary for homotropic cooperativity, are not sufficient to cause homotropic cooperativity since the modified enzyme gave identical perturbations. Interactions of the transition state analog, N-(phosphonacetyl)-L-aspartate, with the modified enzyme were studied. Enzyme kinetic data obtained at low aspartate concentrations showed that this transition state analog does not stimulate activity, but rather exhibits the inhibition predicted for the total absence of homotropic cooperative interactions in the modified enzyme. Spectrophotometric titrations of the number of catalytic sites with the transition state analog showed that the modified enzyme and its isolated subunits possess, respectively, four and two high affinity sites for the inhibitor instead of six and three observed in the case of the normal enzyme and its isolated catalytic subunits. These results are correlated with the lower specific enzymatic activities of the modified enzyme and its catalytic subunits compared to the normal corresponding enzymatic species.     

Aspartate-beta-semialdehyde dehydrogenase from Escherichia coli. Affinity labeling with the substrate analogue L-2-amino-4-oxo-5-chloropentanoic acid: an example of half-site reactivity

The substrate binding site of aspartate-beta-semialdehyde dehydrogenase from Escherichia coli was studied by affinity labeling with L-2-amino-4-oxo-5-chloropentanoic acid. The substrate analogue irreversibly inactivates the enzyme with pseudo-first-order kinetics and with a half-of-the-sites reactivity. The substrate aspartate beta-semialdehyde protects the enzyme against the inactivation. A single group is labeled at the active site and is concluded to be the side-chain of a histidine residue. The amino acid sequence around the active site residue was established from a peptic digest of the labeled enzyme: Phe-Val-Gly-Gly-Asp-(modified residue)-Thr-Val-Ser.     

Subunit interactions in aspartate transcarbamylase. Characterization of a complex between the catalytic and the regulatory subunits.

The complex formed when excess regulatory subunits (r2) of aspartate transcarbamylase is added to a dilute solution of the catalytic subunit (c3) has been further studied. By stabilizing the complex with saturating levels or r2, it was possible to perform ultracentrifugation in sucrose density gradients. The sedimentation coefficient of the complex (7.7 plus or minus 0.2 S) is intermediate between those of the catalytic subunit (5.8 S) and of the native enzyme (11.7 S). Consideration of the likely hydrodynamic properties of the complex suggests that this sedimentation coefficient may be consistent with the c3r6 structure previously proposed. The formation of c3r6 from c3 and r2 is readily reversible. At nonsaturating levels or r2, conversion to the native enzyme (c3r6) takes place. This conversion is inhibited by high concentrations of r2. The c3r6 complex shows Michaelis-Menten kinetics with a low Km for aspartate and considerable substrate inhibition. The pH activity profile at high aspartate concentrations is almost identical with that of the native enzyme. All of these observations suggest that c3r6 represents the relaxed (R) state of aspartate transcarbamylase. The insensitivity of c3r6 toward CTP or ATP can also be explained by considering c3r6 as a stabilized relaxed state. These properties of c3r6 have significant implications regarding the allosteric mechanism of the native enzyme.     

Modulation of phosphofructokinase behavior by chemical modifications during the immobilization process

Phosphofructokinase was immobilized within a protein membrane or on soluble protein polymers using glutaraldehyde as cross-linking reagent. The native enzyme was also modified chemically, using the cross-linking reagent alone. A comparative kinetic investigation of these preparations was carried out. The catalytic activity of the chemically modified enzyme and its affinity towards fructose 6-phosphate decreased significantly; the modified enzyme lost its cooperative properties and the allosteric regulation by AMP was affected. When the chemical treatment was performed in the presence of effectors (AMP or ATP) the allosteric transition induced by AMP was restored, suggesting that the cross-linking reagent modified the AMP regulatory sites, albeit no higher-substrate-affinity enzyme conformation was frozen. Molecular data showed that glutaraldehyde produced intramolecular then intermolecular bonds as its concentration increased. When the enzyme was immobilized into protein membranes or on soluble polymers, the enzyme behavior was quite similar: decrease of affinity towards fructose 6-phosphate but no changes in cooperative properties and modifications of allosteric transition induced by AMP. When AMP was present during the immobilisation process, the enzyme immobilized in this way was no longer sensitive to effectors, either AMP or ATP. It showed Michaelian behavior and higher substrate affinity quite similar to that of the native enzyme. The data suggested that a higher-substrate-affinity enzymatic form was most probably stabilized by immobilization.     

Cooperativity and high pressure: stabilization of the R conformation of the allosteric aspartate transcarbamylase under the influence of pressure.

The allosteric enzyme aspartate transcarbamylase (ATCase) from E. coli shows homotropic cooperative interactions between its six catalytic sites for the binding of the substrate aspartate. This cooperativity is explained by the transition of the enzyme from a conformation which has a low affinity for aspartate (T state) to a conformation with high affinity (R state). The crystallographic structures of these two conformations are known to a resolution of 2.5 A and 2.1 A, respectively, and they reveal an important difference in the quaternary structure of the protein. Enzyme kinetics under high pressure were used to study the transition between the two states. It appears that in the presence of a low concentration of aspartate, conditions under which the enzyme is essentially in the T state, pressure promotes the transition to the R state, the maximal effect being observed at 120 MPa. This transition is accompagnied by a significant deltaV. This observation is in accordance with the change in the protein surface exposed to the solvent, and with the increased number of water molecules bound to the protein. Since the partial specific volume of the enzyme does not change significantly during the T to R transition, the negative deltaV is only related to the change in hydration of the protein. This result emphasizes a significant role of the protein-solvent interactions in this important regulatory conformational change.     

The contribution of individual interchain interactions to the stabilization of the T and R states of Escherichia coli aspartate transcarbamoylase

Stabilization of the T and R allosteric states of Escherichia coli aspartate transcarbamoylase is governed by specific intra- and interchain interactions. The six interchain interactions between Glu-239 in one catalytic chain of one catalytic trimer with both Lys-164 and Tyr-165 of a different catalytic chain in the other catalytic trimer have been shown to be involved in the stabilization of the T state. In this study a series of hybrid versions of aspartate transcarbamoylase was studied to determine the minimum number of these Glu-239 interactions necessary to maintain homotropic cooperativity and the T allosteric state. Hybrids with zero, one, and two Glu-239 stabilizing interactions do not exhibit cooperativity, whereas the hybrids with three or more Glu-239 stabilizing interactions exhibit cooperativity. The hybrid enzymes with one or more of the Glu-239 stabilizing interactions also exhibit heterotropic interactions. Two hybrids with three Glu-239 stabilizing interactions, in different geometric relationships, had identical properties. From this and previous studies, it is concluded that the 239 stabilizing interactions play a critical role in the manifestation of homotropic cooperativity in aspartate transcarbamoylase by the stabilization of the T state of the enzyme. As substrate binding energy is utilized, more and more of the T state stabilizing interactions are relaxed, and finally the enzyme shifts to the R state. In the case of the Glu-239 stabilizing interactions more than three of the interactions must be broken before the enzyme shifts to the R state. The interactions between the catalytic and regulatory chains and between the two catalytic trimers of aspartate transcarbamoylase provide a global set of interlocking interactions that stabilize the T and R states of the enzyme. The substrate-induced local conformational changes observed in the structure of the isolated catalytic subunit drive the quaternary T to R transition of aspartate transcarbamoylase and functionally induced homotropic cooperativity.     

Affinity labeling of the allosteric site of fructose 1,6-bisphosphatase with an AMP analog

D-Fructose 1,6-bisphosphatase [EC 3.1.3.11, FBPase] is one of the key enzymes in glyconeogenesis and its activity is controlled by various effectors such as substrate, AMP and ATP. To analyze this complex regulation system, we tried an affinity labeling of FBPase with an AMP derivative, since AMP is a potent allosteric inhibitor of this enzyme. The results obtained are as follows. 1. To determine the functional groups which are essential for AMP as an inhibitor, inhibitory activities of some AMP derivatives were examined. These derivatives modified at the purine ring or phosphate group lost the activity while one modified at the ribose ring retained the ability to inhibit FBPase. This shows that an affinity labeling reagent should be an AMP derivative in which the ribose ring is modified. 2. 2',3'-Dialdehyde AMP (dial-AMP) was prepared by periodate oxidation of AMP and was reacted with FBPase. Under appropriate conditions, 1 mol of the reagent was incorporated per mol of enzyme subunit with a concomitant loss of enzyme activity. The reaction was prevented by the presence of AMP but not of ATP. The heat-stability, the kinetic parameters and the UV-absorption spectrum of the modified enzyme were all the same as those of native FBPase in the presence of AMP. Thus it was concluded that the allosteric AMP site in FBPase was modified specifically.     

Affinity labeling of pyridoxal kinase with adenosine polyphosphopyridoxal

Pyridoxal kinase is inactivated by preincubation with the affinity label reagent adenosine tetraphosphate pyridoxal (AP4-PL) at a mixing molar ratio of 5:1 AP4-PL contains structural features of the substrates pyridoxal and ATP. The substrate ATP affords substantial protection against inactivation. The extent of chemical modification by the affinity label was determined by measuring the spectroscopic properties of AP4-pyridoxyl chromophores attached to the enzyme after reduction with NaBH4. The incorporation of 2 mol of the affinity label per enzyme dimer is needed for complete inactivation of the kinase. After chymotryptic digestion of the enzyme modified with AP4-PL and reduced with tritiated NaBH4, only one radioactive peptide absorbing at 325 nm was separated by reverse-phase high performance liquid chromatography. The amino acid sequence of the radioactive peptide, elucidated by Edman degradation, revealed that a specific lysyl residue of monomeric pyridoxal kinase has reacted with the affinity label reagent. It is postulated that the modified lysyl residue is involved in direct interactions with phosphoryl groups of ATP.     

Investigation of the active site of the extracellular beta-D-xylosidase from Aspergillus carbonarius

The catalytic amino acid residues of the extracellular beta-D-xylosidase (beta-D-xyloside xylohydrolase, EC 3.2.1.37) from Aspergillus carbonarius was investigated by the pH dependence of reaction kinetic parameters and chemical modifications of the enzyme. The pH dependence curves gave apparent pK values of 2.7 and 6.4 for the free enzyme, while pK value of 4.0 was obtained for the enzyme-substrate complex using p-nitrophenyl beta-D-xyloside as a substrate. These results suggested that a carboxylate group and a protonated group--presumably a histidine residue--took part in the binding of the substrate but only a carboxylate group was essential in the substrate cleavage. Carbodiimide- and Woodward's reagent K-mediated chemical modifications of the enzyme also supported that a carboxylate residue, located in the active center, was fundamental in the catalysis. The pH dependence of inactivation revealed the involvement of a group with pK value of 4.4, proving that a carboxylate residue relevant for hydrolysis was modified. During modification V(max) decreased to 10% of that of the unmodified enzyme and K(m) remained unchanged, supporting that the modified carboxylate group participated in the cleavage and not in the binding of the substrate. We synthesized and tested a new, potential affinity label, N-bromoacetyl-beta-d-xylopyranosylamine for beta-D-xylosidase. The A. carbonarius beta-D-xylosidase was irreversible inactivated by N-bromoacetyl-beta-D-xylopyranosylamine. The competitive inhibitor beta-D-xylopyranosyl azide protected the enzyme from inactivation proving that the inactivation took place in the active center. Kinetic analysis indicated that one molecule of reagent was necessary for inactivation of one molecule of the enzyme.     

Functionally important arginine residues of aspartate transcarbamylase.

The reaction of phenylglyoxal with aspartate transcarbamylase and its isolated catalytic subunit results in complete loss of enzymatic activity (Kantrowitz, E. R., and Lipscomb, W. N. (1976) J. Biol. Chem. 251, 2688-2695). If N-(phosphonacetyl)-L-aspartate is used to protect the active site, we find that phenylglyoxal causes destruction of the enzyme's susceptibility to activation by ATP and inhibition by CTP. Furthermore, CTP only minimally protects the regulatory site from reaction with this reagent. The modified enzyme still binds CTP although with reduced affinity. After reaction with phenylglyoxal, the native enzyme shows reduced cooperativity. The hybrid with modified regulatory subunits and native catalytic subunits exhibits slight heterotropic or homotropic properties, while the reverse hybrid, with modified catalytic subunits and native regulatory subunits, shows much reduced homotropic properties but practically normal heterotropic interactions. The decrease in the ability of CTP to inhibit the enzyme correlates with the loss of 2 arginine residues/regulatory chain (Mr = 17,000). Under these reaction conditions, 1 arginine residue is also modified on each catalytic chain (Mr = 33,000). Reaction rate studies of p-hydroxymercuribenzoate, with the liganded and unliganded modified enzyme suggest that the reaction with phenylglyoxal locks the enzyme into the liganded conformation. The conformational state of the regulatory subunit is implicated as having a critical role in the expression of the enzyme's heterotropic and homotropic properties.     

Affinity labeling of a subsite of Taka-amylase A by the fluorescent reagent o-phthalaldehyde

A cross-linked modification of Lys residue located at the subsite of the enzyme active site of Taka-amylase A was attained by the use of the fluorescent reagent of o-phthalaldehyde (OPA). The fluorescence and uv absorption at 337 nm derived from the isoindole ring, which was produced by cross-linking through the epsilon-amino group of Lys and the thiol group of the Cys residue, provided the evidence for the OPA-mediated inactivation of Taka-amylase A. Kinetic analysis showed that 1 mol of OPA per mole of enzyme was incorporated, which corresponded closely with the value obtained by the uv absorption. Because the OPA inactivation was retarded by the substrate analog of alpha-cyclodextrin, OPA modification was classified as a type of affinity labeling reaction. A remarkable increase in the pI value from 4.0 to 5.6 upon the modification led to clear separation of the modified enzyme from the native Taka-amylase A by a DEAE-Sephacel column and led to the charge isomer pattern on gel electrophoresis performed according to the method of Hedrick and Smith. Moreover, the affinity gel electrophoresis showed that the modified enzyme completely lost the affinity for the substrate soluble starch, which indicated that the subsite modification occurred.     

Coupling of homotropic and heterotropic interactions in Escherichia coli aspartate transcarbamylase

Several types of conditions allow the disconnection of homotropic and heterotropic interactions in Escherichia coli aspartate transcarbamylase. A model that includes a concerted gross conformational change corresponding to the homotropic cooperative interactions between the catalytic sites and local “site by site” effects promoted by the effectors accounts for this disconnection as well as for the other known properties of the enzyme. However, the substrate concentration influences the extent of stimulation and feedback inhibition of the catalytic activity by the effectors. This result is explained by assuming that these effectors promote a “primary effect”, which is exerted locally “site by site”, and a “secondary effect”, which is mediated by the substrate. As predicted by the model, relaxed (R) forms of the enzyme show only the primary effect. In addition 2-ThioU-aspartate transcarbamylase, a modified form of the enzyme in which the homotropic cooperative interactions between the catalytic sites are selectively abolished, shows the same heterogeneity in CTP binding sites as normal aspartate transcarbamylase.     

Evidence for an arginine residue at the substrate binding site of Escherichia coli adenylosuccinate synthetase as studied by chemical modification and site-directed mutagenesis

Chemical modification of adenylosuccinate synthetase from Escherichia coli with phenylglyoxal resulted in an inhibition of enzyme activity with a second-order rate constant of 13.6 M-1 min-1. The substrates, GTP or IMP, partially protected the enzyme against inactivation by the chemical modification. The other substrate, aspartate, had no such effect even at a high concentration. In the presence of both IMP and GTP during the modification, nearly complete protection of the enzyme against inactivation was observed. Stoichiometry studies with [7-14C]phenylglyoxal showed that only 1 reactive arginine residue was modified by the chemical reagent and that this arginine residue could be shielded by GTP and IMP. Sequence analysis of tryptic peptides indicated that Arg147 is the site of phenylglyoxal chemical modification. This arginine has been changed to leucine by site-directed mutagenesis. The mutant enzyme (R147L) showed increased Michaelis constants for IMP and GTP relative to the wild-type system, whereas the Km for aspartate exhibited a modest decrease as compared with the native enzyme. In addition, kcat of the R147L mutant decreased by a factor of 1.3 x 10(4). On the bases of these observations, it is suggested that Arg147 is critical for enzyme catalysis.     

Picosecond dynamics of T and R forms of aspartate transcarbamylase: a neutron scattering study

E. coli aspartate transcarbamylase (ATCase) is a 310 kDa allosteric enzyme which catalyses the first committed step in pyrimidine biosynthesis. The binding of its substrates, carbamylphosphate and aspartate, induces significant conformational changes. This enzyme shows homotropic cooperative interactions between the catalytic sites for the binding of aspartate. This property is explained by a quaternary structure transition from T state (aspartate low affinity) to R state (aspartate high affinity) accompanied by a 5% increase of radius of gyration of ATCase. The same quaternary structure change is observed upon binding of the bisubstrate analogue PALA (N-(phosphonacetyl)-L-aspartate. Owing to the large incoherent neutron scattering cross-section of the hydrogen atom and the abundance of this element in proteins, inelastic neutron scattering gives a global view of protein dynamics as sensed via the individual motions of its hydrogen atoms. We present neutron scattering results of the local dynamics (few angstroms), at short time (few tens of picoseconds), of ATCase in T and R forms. Compared to the T form, we observe an increased mobility of the protein in the R form that we associate to an increase of accessible surface area to the solvent. Beyond this specific result, this highlights the key role of the accessible surface area (ASA) in dynamic contribution to inelastic neutron data in the picosecond time scale. In particular, we want to stress out (i) that a difference at the picosecond time scale does not allow to conclude to a difference in the dynamics at a longer time scale and to address whether the T state is looser than the R state (ii) how challenging is, any comparison in terms of general dynamics (tense or relaxed) between dynamic values deduced from experimental neutron data on proteins with different sequences and therefore ASA. This caveat holds particularly when comparing dynamics of a mesophile with the corresponding extremophile.     

Affinity labeling of catalytic subunit of bovine heart muscle cyclic AMP-dependent protein kinase by 5'-p-fluorosulfonylbenzoyladenosine.

Incubation of 5'-p-fluorosulfonylbenzoyladenosine with the catalytic subunit of bovine cardiac muscle cyclic AMP-dependent protein kinase led to the formation of an inactive enzyme irreversibly modified with approximately one mol of reagent per mol of subunit. The inactivation reaction followed pseudofirst order kinetics. The rate of inactivation at various reagent concentrations exhibited saturation kinetics implying that the reagent reversibly binds to the enzyme prior to inactivation. The addition of MgATP, MgADP, or MgAMP-PNP to the reaction mixture fully protected the enzyme from inactivation by 5'-p-fluorosulfonylbenzoyladenosine. The reagent was demonstrated to be a competitive inhibitor of MgATP with a Ki of 0.235 mM. Metal-free nucleotides were without effect upon the reaction rate while metal ions alone accelerated the inactivation rate up to 7-fold. The inclusion of casein or synthetic peptide substrate in the incubation mixture did not affect the reaction kinetics. Reaction of 5'-p-fluorosulfonylbenzoyladenosine with the kinase subunit exhibits all of the characteristics of affinity labeling of the MgATP-binding site.     

Three of the six possible intersubunit stabilizing interactions involving Glu-239 are sufficient for restoration of the homotropic and heterotropic properties of Escherichia coli aspartate transcarbamoylase

A hybrid version of Escherichia coli aspartate transcarbamoylase was investigated in which one catalytic subunit has the wild-type sequence, and the other catalytic subunit has Glu-239 replaced by Gln. Since Glu-239 is involved in intersubunit interactions, this hybrid could be used to evaluate the extent to which T state stabilization is required for homotropic cooperativity and for heterotropic effects. Reconstitution of the hybrid holoenzyme (two different catalytic subunits with three wild-type regulatory subunits) was followed by separation of the mixture by anion-exchange chromatography. To make possible the resolution of the three holoenzyme species formed by the reconstitution, the charge of one of the catalytic subunits was altered by the addition of six aspartic acid residues to the C terminus of each of the catalytic chains (AT-C catalytic subunit). Control experiments indicated that the AT-C catalytic subunit as well as the holoenzyme formed with AT-C and wild-type regulatory subunits had essentially the same homotropic and heterotropic properties as the native catalytic subunit and holoenzyme, indicating that the addition of the aspartate tail did not influence the function of either enzyme. The control reconstituted holoenzyme, in which both catalytic subunits have Glu-239 replaced by Gln, exhibited no cooperativity, an enhanced affinity for aspartate, and essentially no heterotropic response identical to the enzyme isolated without reconstitution. The hybrid containing one normal and one mutant catalytic subunit exhibited homotropic cooperativity with a Hill coefficient of 1.4 and responded to the nucleotide effectors at about 50% of the level of the wild-type enzyme. Small angle x-ray scattering experiments with the hybrid enzyme indicated that in the absence of ligands it was structurally similar, but not identical, to the T state of the wild-type enzyme. In contrast to the wild-type enzyme, addition of carbamoyl phosphate induced a significant alteration in the scattering pattern, whereas the bisubstrate analog N-phosphonoacetyl-L-aspartate induced a significant change in the scattering pattern indicating the transition to the R-structural state. These data indicate that in the hybrid enzyme only three of the usual six interchain interactions involving Glu-239 are sufficient to stabilize the enzyme in a low affinity, low activity state and allow an allosteric transition to occur.     

Cocrystallization of a mutant aspartate aminotransferase with a C5-dicarboxylic substrate analog: structural comparison with the enzyme-C4-dicarboxylic analog complex

A mutant Escherichia coil aspartate aminotransferase with 17 amino acid substitutions (ATB17), previously created by directed evolution, shows increased activity for beta-branched amino acids and decreased activity for the native substrates, aspartate and glutamate. A new mutant (ATBSN) was generated by changing two of the 17 mutated residues back to the original ones. ATBSN recovered the activities for aspartate and glutamate to the level of the wild-type enzyme while maintaining the enhanced activity of ATB17 for the other amino acid substrates. The absorption spectrum of the bound coenzyme, pyridoxal 5'-phosphate, also returned to the original state. ATBSN shows significantly increased affinity for substrate analogs including succinate and glutarate, analogs of aspartate and glutamate, respectively. Hence, we could cocrystallize ATBSN with succinate or glutarate, and the structures show how the enzyme can bind two kinds of dicarboxylic substrates with different chain lengths. The present results may also provide an insight into the long-standing controversies regarding the mode of binding of glutamate to the wild-type enzyme.     

The catalytic site of Escherichia coli aspartate transcarbamylase: interaction between histidine 134 and the carbonyl group of the substrate carbamyl phosphate

Previous pKa determinations indicated that histidine 134, present in the catalytic site of aspartate transcarbamylase, might be the group involved in the binding of the substrate carbamyl phosphate and, possibly, in the catalytic efficiency of this enzyme. In the present work, this residue was replaced by an asparagine through site-directed mutagenesis. The results obtained show that histidine 134 is indeed the group of the enzyme whose deprotonation increases the affinity of the catalytic site for carbamyl phosphate. In the wild-type enzyme this group can be titrated only by those carbamyl phosphate analogues that bear the carbonyl group. In the modified enzyme the group whose deprotonation increases the catalytic efficiency is still present, indicating that this group is not the imidazole ring of histidine 134 (pKa = 6.3). In addition, the pKa of the still unknown group involved in aspartate binding is shifted by one unit in the mutant as compared to the wild type.     

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.