An essential residue at the active site of aspartate transcarbamylase.

Reaction of phenylglyoxal with aspartate transcarbamylase and its isolated catalytic subunit results in complete loss of enzymatic activity. This modification reaction is markedly influenced by pH and is partially reversible upon dialysis. Carbamyl phosphate or carbamyl phosphate with succinate partially protect the catalytic subunit and the native enzyme from inactivation by phenylglyoxal. In the native enzyme complete protection from inactivation is afforded by N-(phosphonacetyl)-L-aspartate. The decrease in enzymatic activity correlates with the modification of 6 arginine residues on each aspartate transcarbamylase molecule, i.e. 1 arginine per catalytic site. The data suggest that the essential arginine is involved in the binding of carbamyl phosphate to the enzyme. Reaction of the single thiol on the catalytic chain with 2-chloromercuri-4-nitrophenol does not prevent subsequent reaction with phenylglyoxal. If N-(phosphonacetyl)-L-aspartate is used to protect the active site we find that phenylglyoxal also causes the loss of activation of ATP and inhibition by CTP. The rate of loss of heterotropic effects is exactly the same for both nucleotides indicating that the two opposite regulatory effects originate at the same location on the enzyme, or are transmitted by the same mechanism between the subunits, or both.     

Communication between polypeptide chains in aspartate transcarbamoylase. Conformational changes at the active sites of unliganded chains resulting from ligand binding to other chains

A largely inactive derivative of the catalytic subunit of Escherichia coli aspartate transcarbamoylase containing trinitrophenyl groups on lysine 83 and 84 was used to study communication between polypeptide chains in the holoenzyme and the isolated catalytic trimers. Addition of native regulatory dimers to the derivative yielded a holoenzyme-like complex of low activity which exhibited sigmoidal kinetics and was inhibited by CTP and activated by ATP. The binding of CTP and ATP to the regulatory subunits caused significant and opposite changes in the absorption spectrum resulting from changes in the environment of the sensitive chromophores at the active sites. In allosteric hybrid molecules containing one native and one trinitrophenylated catalytic subunit, along with native regulatory subunits, the binding of a bisubstrate analog, N-(phosphonacetyl)-L-aspartate, to the native catalytic subunit resulted in a perturbation of the spectrum of the chromophore on the unliganded modified chains. Thus the conformational changes associated with the allosteric transition responsible for both heterotropic and homotropic effects are propagated from the sites of ligand binding to the active sites of unliganded distant chains. In addition to the communication from regulatory chains to catalytic chains and the cross-talk from one catalytic subunit to the other, communication between individual catalytic chains in isolated trimers was also demonstrated. By constructing hybrid trimers containing one trinitrophenylated chain and two native chains, we could detect a change in the environment of the chromophore upon the binding of the bisubstrate analog to the native chains.     

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.     

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.     

Discrimination between nucleotide effector responses of aspartate transcarbamoylase due to a single site substitution in the allosteric binding site

The substitution of alanine for lysine at position 56 of the regulatory polypeptide of aspartate transcarbamoylase affected both homotropic and heterotropic characteristics. In the absence of effectors, the ALAr56-substituted holoenzyme lost the homotropic cooperativity observed for aspartate in the wild-type holoenzyme. Under conditions of allosteric inhibition in the presence of 2mM CTP, the cooperative character of ATCase was restored, and the Hill coefficient increased from 1.0 to 1.7. In contrast to the native enzyme, the altered enzyme did not respond to ATP; however, ATP could still bind to the enzyme as demonstrated by its direct competition with CTP. Furthermore, the recently observed CTP-UTP synergism of the wild-type enzyme was not detectable. The site-directed mutant enzyme could not be activated by low levels of the bisubstrate analogue, N-(phosphonacetyl)-L-aspartate, and the rate of association of pHMB with the cysteine residues located at the interface of the catalytic and regulatory chains was slightly altered. These characteristics suggested that the mutant holoenzyme assumed a relaxed (or abnormal T state) conformation. Thus, this single substitution differentially affected the heterotropic responses to the various allosteric effectors of ATCase and eliminated the homotropic characteristics in response to aspartate in the absence of CTP.     

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.     

Regulation of catalytic activity of a multifunctional polyketide biosynthetic enzyme, 6-hydroxymellein synthase, by interaction between NADPH and phenylglyoxal-sensitive amino acid residue at the reaction center

Treatment of 6-hydroxymellein synthase, a multifunctional polyketide biosynthetic enzyme in carrot cells, with phenylglyoxal yielded a chemically modified protein in which approximately two moles of the reagent were covalently attached to each subunit of the enzyme. Only NADH- but not NADPH-associated form of native 6-hydroxymellein synthase was inhibited by cerulenin; however, the NADPH-synthase complex lost the insensitivity by the chemical modification of the enzyme protein with phenylglyoxal. Appreciable differences in K(m) values observed between the NADPH- and NADH-associated enzymes were greatly reduced by the treatment with phenylglyoxal. Although the catalytic activity of the NADPH-associated synthase was enhanced by the addition of free CoA, the compound exhibited a significant inhibitory activity to the phenylglyoxal-modified enzyme. A marked deuterium isotope effect in the catalytic reaction of the native synthase-NADPH complex was appreciably decreased in the chemically modified enzyme. These results strongly suggest that an electrostatic interaction between the phosphate group attached to the 2'-position of adenosyl moiety of NADPH and the phenylglyoxal-sensitive amino acid residue, probably arginine, at the reaction center of 6-hydroxymellein synthase regulates several biochemical properties of this multifunctional enzyme.     

Comparison of the aspartate transcarbamoylases from Serratia marcescens and Escherichia coli

The aspartate transcarbamoylases (ATCase, EC 2.1.3.2) of Escherichia coli and Serratia marcescens have similar dodecameric enzyme structures (2(c3):3(r2] but differ in both regulatory and catalytic characteristics. The catalytic cistrons (pyrB) of the ATCases from E. coli and S. marcescens encode polypeptides of 311 and 306 amino acids, respectively; there is a 76% identity between the DNA sequences and an overall amino acid homology of 88% (38 differences). The regulatory cistrons (pyrI) of these ATCases encode polypeptides of 153 and 154 amino acids, respectively, and there is a 75% identity between the DNA sequences and an overall amino acid homology of 77% (36 differences). In both species, the two genes are arranged as a bicistronic operon, with pyrB promoter proximal. A comparison of the deduced amino acid sequences reveals that the active site and the allosteric binding sites, as well as most of the intrasubunit interactions and intersubunit associations, are conserved in the E. coli and the S. marcescens enzymes; however, there are specific differences which undoubtedly contribute to the catalytic and regulatory differences between the enzymes of the two species. These differences include residues that have been implicated in the T-R transition, c1:r1 interface interactions, and the CTP binding site. A hybrid ATCase assembled in vivo with catalytic subunits from E. coli and regulatory subunits from S. marcescens has a 6 mM requirement for aspartate at half-maximal saturation, similar to the 5.5 mM aspartate requirement of the native E. coli holoenzyme at half-maximal saturation. However, the heterotropic response of this hybrid enzyme is characteristic of the heterotropic response of the native S. marcescens holoenzyme: ATP activation and CTP activation. Activation by both allosteric effectors indicates that the heterotropic response of this hybrid holoenzyme (Cec:Rsm) is determined by the associated S. marcescens regulatory subunits.     

Importance of domain closure for the catalysis and regulation of Escherichia coli aspartate transcarbamoylase

Two hybrid versions of Escherichia coli aspartate transcarbamoylase were studied to determine the influence of domain closure on the homotropic and heterotropic properties of the enzyme. Each hybrid holoenzyme had one wild-type and one inactive catalytic subunit. In the first case the inactive catalytic subunit had Arg-54 replaced by alanine. The holoenzyme with this mutation in all six catalytic chains exhibits a 17,000-fold reduction in activity, no loss in substrate affinity, and an R state structurally identical to that of the wild-type enzyme. In the second case, the inactive catalytic subunit had Arg-105 replaced by alanine. The holoenzyme with this mutation in all six catalytic chains exhibits a 1,100-fold reduction in activity, substantial loss in substrate affinity, and loss of the ability to be converted to the R state. Thus, the R54A substitution results in a holoenzyme that can undergo closure of the catalytic chain domains to form the high activity, high affinity active site and to undergo the allosteric transition, whereas the R105A substitution results in a holoenzyme that can neither undergo domain closure nor the allosteric transition. The hybrid holoenzyme with one wild-type and one R54A catalytic subunit exhibited the same maximal velocity per active site as the wild-type holoenzyme, reduced cooperativity, and normal heterotropic interactions. The hybrid with one wild-type and one R105A catalytic subunit exhibited significantly reduced maximal velocity per active site as compared with the wild-type holoenzyme, reduced cooperativity, and substantially reduced heterotropic interactions. Small angle x-ray scattered was used to verify that the R105A-containing hybrid could attain an R state structure. These results indicate the global nature of the conformational changes associated with the allosteric transition in the enzyme. If one catalytic subunit cannot undergo domain closure to create the active sites, then the entire molecule cannot attain the high activity, high activity R state.     

An aspartate transcarbamylase lacking catalytic subunit interactions. II. Regulatory subunits are responsible for the lack of co-operative interactions between catalytic sites. Drastic feedback inhibition does not restore these interactions

It has been previously reported (Kerbiriou &; Hervé, 1972) that, when a uracil-requiring mutant of Escherichia coli is derepressed for the biosynthesis of the enzymes of the pyrimidine pathway in the presence of 2-thiouracil, it synthesizes a modified aspartate transcarbamylase which is still sensitive to the feedback inhibitor CTP, but which does not show homotropic positive interactions between catalytic sites. It is shown here that these homotropic interactions do not reappear upon strong inhibition by CTP, indicating that the two types of interactions are really disconnected and must involve different molecular mechanisms. CTP is acting at the level of the apparent K_m of the enzyme for aspartate. It is also the case for ATP, which stimulates 2-thiouracil aspartate-transcarbamylase. Kinetic studies of the hybrid molecules made up of subunits prepared from normal and modified enzymes show that it is a modification at the level of the regulatory subunits which is responsible for the lack of co-operative interactions between catalytic sites. These results are discussed in terms of a four-state model.     

Interaction of gamma-glutamyl transpeptidase with glutathione involves specific arginine and lysine residues of the heavy subunit.

Gamma-glutamyl transpeptidase, an enzyme of importance in glutathione metabolism, consists of two subunits, one of which (the light subunit, Mr 22,000; residues 380-568; rat kidney) contains residue Thr-523, which selectively interacts with the substrate analog acivicin to form an adduct that is apparently analogous to the gamma-glutamyl enzyme intermediate formed in the normal reaction (Stole, E., Seddon, A. P., Wellner, D., and Meister, A. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 1706-1709). The present studies indicate that specific arginine and lysine residues of the heavy subunit (Mr 51,000; residues 31-379) participate in catalysis by binding the substrates. Selective labeling studies of the enzyme with [14C]phenylglyoxal showed that Lys-99 and Arg-111 were modified. This appears to be the first instance in which phenylglyoxal was found to react with an enzyme lysine residue. Incorporation of [14C]phenylglyoxal into Lys-99 was decreased in the presence of acceptor site selective compounds. Incorporation into both Lys-99 and Arg-111 was decreased in the presence of glutathione. The findings suggest that Lys-99 and Arg-111 interact, respectively, with the omega- and alpha-carboxyl groups of glutathione. That these putative electrostatic binding sites are on the heavy subunit indicates that both subunits contribute to the active center. Two additional heavy subunit arginine residues become accessible to modification by phenylglyoxal when acivicin is bound, suggesting that interaction with acivicin is associated with a conformational change.     

Divergent allosteric patterns verify the regulatory paradigm for aspartate transcarbamylase

The native Escherichia coli aspartate transcarbamoylase (ATCase, E.C. 2.1.3.2) provides a classic allosteric model for the feedback inhibition of a biosynthetic pathway by its end products. Both E. coli and Erwinia herbicola possess ATCase holoenzymes which are dodecameric (2(c3):3(r2)) with 311 amino acid residues per catalytic monomer and 153 and 154 amino acid residues per regulatory (r) monomer, respectively. While the quaternary structures of the two enzymes are identical, the primary amino acid sequences have diverged by 14 % in the catalytic polypeptide and 20 % in the regulatory polypeptide. The amino acids proposed to be directly involved in the active site and nucleotide binding site are strictly conserved between the two enzymes; nonetheless, the two enzymes differ in their catalytic and regulatory characteristics. The E. coli enzyme has sigmoidal substrate binding with activation by ATP, and inhibition by CTP, while the E. herbicola enzyme has apparent first order kinetics at low substrate concentrations in the absence of allosteric ligands, no ATP activation and only slight CTP inhibition. In an apparently important and highly conserved characteristic, CTP and UTP impose strong synergistic inhibition on both enzymes. The co-operative binding of aspartate in the E. coli enzyme is correlated with a T-to-R conformational transition which appears to be greatly reduced in the E. herbicola enzyme, although the addition of inhibitory heterotropic ligands (CTP or CTP+UTP) re-establishes co-operative saturation kinetics. Hybrid holoenzymes assembled in vivo with catalytic subunits from E. herbicola and regulatory subunits from E. coli mimick the allosteric response of the native E. coli holoenzyme and exhibit ATP activation. The reverse hybrid, regulatory subunits from E. herbicola and catalytic subunits from E. coli, exhibited no response to ATP. The conserved structure and diverged functional characteristics of the E. herbicola enzyme provides an opportunity for a new evaluation of the common paradigm involving allosteric control of ATCase.     

Structure-function relationship in allosteric aspartate carbamoyltransferase from Escherichia coli. II. Involvement of the C-terminal region of the regulatory chain in homotropic and heterotropic interactions

The modified aspartate transcarbamylase (ATCase) encoded by the transducing phage described by Cunin et al. has been purified to homogeneity. In this altered form of enzyme (pAR5-ATCase) the last eight amino acids of the C-terminal end of the regulatory chains are replaced by a sequence of six amino acids coded for by the lambda DNA. This modification has very informative consequences on the allosteric properties of ATCase. pAR5-ATCase lacks the homotropic co-operative interactions between the catalytic sites for aspartate binding and is "frozen" in the R state. In addition, this altered form of enzyme is insensitive to the physiological feedback inhibitor CTP, in spite of the fact that this nucleotide binds normally to the regulatory sites. Conversely, pAR5-ATCase is fully sensitive to the activator ATP. However, this activation is limited to the extent of the previously described "primary effect" as expected from an ATCase form "frozen" in the R state. These results emphasize the importance of the three-dimensional structure of the C-terminal region of the regulatory chains for both homotropic and heterotropic interactions. In addition, they indicate that the primary effects of CTP and ATP involve different features of the regulatory chain-catalytic chain interaction area.     

Different amino acid substitutions at the same position in the nucleotide-binding site of aspartate transcarbamoylase have diverse effects on the allosteric properties of the enzyme

Site-directed mutagenesis was used to determine how the allosteric properties of aspartate transcarbamoylase (ATCase) are affected by amino acid replacements in the nucleotide binding region of the regulatory polypeptide chains. Amino acid substitutions were made for both Lys-60 and Lys-94 in the regulatory chain since those residues have been implicated by x-ray diffraction studies, chemical modification experiments, and site-directed mutagenesis as playing a role in binding CTP and ATP. Lys-60 was replaced by His, Arg, Gln, and Ala, and Lys-94 was changed to His. These mutant forms of ATCase exhibit bewildering changes in the allosteric properties compared to the wild-type enzyme as well as altered affinities for the nucleotide effectors. The enzyme containing His-60 lacks both homotropic and heterotropic effects and exhibits no detectable binding of nucleotides. In contrast, the holoenzymes containing either Gln-60 or Arg-60 retain both homotropic and heterotropic effects. Replacement of Lys-60 by Ala yields a derivative exhibiting altered heterotropic effects involving insensitivity to CTP and activation by ATP. The mutant enzyme containing His-94 in place of Lys exhibits cooperativity with reduced affinity for nucleotides. The multiple substitutions at Lys-60 in the nucleotide binding region of the regulatory chains of ATCase demonstrate that different amino acids in the same location can alter indirectly the delicate balance of interactions responsible for the allosteric properties of ATCase. The studies show that it is hazardous and frequently unwarranted from single amino acid replacements of a specific residue to attribute to that residue the properties observed for the wild-type enzyme.     

Asymmetry of binding and physical assignments of CTP and ATP sites in aspartate transcarbamoylase.

The allosteric effectors of aspartate transcarbamoylase from Escherichia coli, CTP and ATP, associate with both the regulatory and the catalytic moieties of the enzyme. Studies with isolated, active subunits yield one binding site per regulatory dimer and one per catalytic trimer. Investigations of effector association with hybrid enzymes, containing either the three regulatory dimers or the two catalytic trimers in inactivated forms, indicate that the data obtained with isolated subunits can be used to analyze the binding patterns of these ligands to the native hexamer. Thus, the nonlinear Scatchard plots, characteristic of the binding of CTP and ATP to the native enzyme, can be interpreted in terms of three effector molecules associating with the regulatory subunits, and two binding to the catalytic moiety of the enzyme. Results with native protein in the presence of saturating concentrations of active site ligands support these assignments. The differences between the binding isotherms of CTP and ATP to the enzyme are due to their different affinities to the two types of subunits. The apparent half-of-the-site saturation of the regulatory moiety of aspartate transcarbamoylase supports the concept that this protein has a tendency to exist in an asymmetric state.     

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.     

The importance of the link between Glu204 of the catalytic chain and Arg130 of the regulatory chain for the homotropic and heterotropic properties of Escherichia coli aspartate transcarbamoylase

Recent x-ray crystallographic studies of aspartate transcarbamoylase bound with CTP have detected molecular asymmetry in the interface between the catalytic and regulatory subunits (Kim, K. H., Pan, Z., Honzatko, R. B., Ke, H.-M., and Lipscomb, W. N. (1987) J. Mol. Biol. 196, 863-875). In three of the six interfaces, a salt link occurs between Arg130 of the regulatory chain and Glu204 of the catalytic chain; however, these same residues are 15 A apart in the other three interfaces. In order to determine if this is important for the function of the enzyme, two mutant versions of aspartate transcarbamoylase were created by site-specific mutagenesis. Glu204 of the catalytic chain was converted to a glutamine (Glu204c----Gln) and Arg130 of the regulatory chain was converted to a glycine (Arg130r----Gly). The thermal stability of the Arg130r----Gly enzyme is dramatically reduced, whereas the thermal stability of the Glu204c----Gln enzyme is unaltered compared to the wild-type enzyme. The maximal velocity of both mutant enzymes is identical with that of the wild-type enzyme, however both mutant enzymes have altered substrate affinity and regulatory properties. Based on these studies, the link between Glu204 of the catalytic chain and Arg130 of the regulatory chain is important for the heterotropic properties of the enzyme. Furthermore, the interface between the domain of the regulatory chain which binds zinc and the domain of the catalytic chain which binds aspartate may be more important for CTP inhibition than ATP activation. These data also suggest that heterotropic cooperativity is very sensitive to alterations in the catalytic-regulatory interface. However, no clear relationship has been observed between the structural asymmetry and the function of the enzyme.     

Arginine-specific modification of rabbit muscle phosphoglucose isomerase: Differences in the inactivation by phenylglyoxal and butanedione and in the protection by substrate analogs

Rabbit muscle phosphoglucose isomerase was modified with phenylglyoxal or 2,3-butanedione, the reaction with either reagent resulting in loss of enzymatic activity in a biphasic mode. At slightly alkaline pH butanedione was found to be approximately six times as effective as phenylglyoxal. The inactivation process could not be significantly reversed by removal of the modifier. Competitive inhibitors of the enzyme protected partially against loss of enzyme activity by either modification. The only kind of amino acid residue affected was arginine. However, more than one arginine residue per enzyme subunit was found to be susceptible to modification by the dicarbonyl reagents. From protection experiments it was concluded (i) that both modifiers react specifically with an arginine in the phosphoglucose isomerase active site and nonspecifically with one or more arginine residues elsewhere in the enzyme molecule, (ii) that modification at either loci causes loss of catalytic activity, and (iii) that butanedione has a higher preference for active site arginine than for arginine residues outside of the catalytic center whereas the opposite is true for phenylglyoxal.     

Structure-function relationship in allosteric aspartate carbamoyltransferase from Escherichia coli. I. Primary structure of a pyrI gene encoding a modified regulatory subunit

In a previous article, we have identified a lambda bacteriophage directing the synthesis of a modified aspartate carbamoyltransferase lacking substrate-co-operative interactions and insensitive to the feedback inhibitor CTP. These abnormal properties were ascribed to a mutation in the gene pyrI encoding the regulatory polypeptide chain of the enzyme. We now report the sequence of the mutated pyrI and show that, during the generation of this pyrBI-bearing phage, six codons from lambda DNA have been substituted for the eight terminal codons of the wild-type gene. A model is presented for the formation of this modified pyrI gene during the integrative recombination of the parental lambda phage with the Escherichia coli chromosome. An accompanying paper emphasizes the importance of the carboxy-terminal end of the regulatory chain for the homotropic and heterotropic interactions of aspartate carbamoyltransferase.     

Isolation of a single activating allosteric interaction in phosphofructokinase from Escherichia coli

Escherichia coli phosphofructokinase 1 (EcPFK) is a homotetramer with four active and four allosteric sites. Understanding of the structural basis of allosteric activation of EcPFK by MgADP is complicated by the multiplicity of binding sites. To isolate a single heterotropic allosteric interaction, hybrid tetramers were formed between wild-type and mutant EcPFK subunits in which the binding sites of the mutant subunits have decreased affinity for their respective ligands. The 1:3 (wild-type:mutant) hybrid that contained only one native active site and one native allosteric site was isolated. The affinity for the substrate fructose-6-phosphate (Fru-6-P) of a single wild-type active site is greatly decreased over that displayed by the wild-type tetramer due to the lack of homotropic activation. The free energy of activation by MgADP for this heterotropic interaction is -0.58 kcal/mol at 8.5 degrees C. This compares to -2.87 kcal/mol for a hybrid with no homotropic coupling but all four unique heterotropic interactions. Therefore, the isolated interaction contributes 20% of the total heterotropic coupling. By comparison, wild-type EcPFK exhibits a coupling free energy between Fru-6-P and MgADP of -1.56 kcal/mol under these conditions, indicating that the effects of MgADP are diminished by a homotropic activation equal to -1.3 kcal/mol. These data are not consistent with a concerted allosteric mechanism.