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.     

Exosite interactions determine the affinity of factor X for the extrinsic Xase complex

The initiation of coagulation results from the activation of factor X by an enzyme complex (Xase) composed of the trypsin-like serine proteinase, factor VIIa, bound to tissue factor (TF) on phospholipid membranes. We have investigated the basis for the protein substrate specificity of Xase using TF reconstituted into vesicles of phosphatidylcholine, phosphatidylserine, or pure phosphatidylcholine. We show that occupation of the active site of VIIa within Xase by a reversible inhibitor or an alternate peptidyl substrate is sufficient to exclude substrate interactions at the active site but does not alter the affinity of Xase for factor X. This is evident as classical competitive inhibition of peptidyl substrate cleavage but as classical noncompetitive inhibition of factor X activation by active site-directed ligands. This implies that the productive recognition of factor X by Xase arises from a multistep reaction requiring an initial interaction at sites on the enzyme complex distinct from the active site (exosites), followed by active site interactions and bond cleavage. Exosite interactions determine protein substrate affinity, whereas the second binding step influences the maximum catalytic rate for the reaction. We also show that competitive inhibition can be achieved by interfering with exosite binding using factor X derivatives that are expected to have limited or abrogated interactions with the active site of VIIa within Xase. Thus, substrate interactions at exosites, sites removed from the active site of VIIa within the enzyme complex, determine affinity and binding specificity in the productive recognition of factor X by the VIIa-TF complex. This may represent a prevalent strategy through which distinctive protein substrate specificities are achieved by the homologous enzymes of coagulation.     

The Role of Strong Electrostatic Interactions at the Dimer Interface of Human Glutathione Synthetase

The obligate homodimer human glutathione synthetase (hGS) provides an ideal system for exploring the role of protein–protein interactions in the structural stability, activity and allostery of enzymes. The two active sites of hGS, which are 40 Å apart, display allosteric modulation by the substrate γ-glutamylcysteine (γ-GC) during the synthesis of glutathione, a key cellular antioxidant. The two subunits interact at a relatively small dimer interface dominated by electrostatic interactions between S42, R221, and D24. Alanine scans of these sites result in enzymes with decreased activity, altered γ-GC affinity, and decreased thermal stability. Molecular dynamics simulations indicate these mutations disrupt interchain bonding and impact the tertiary structure of hGS. While the ionic hydrogen bonds and salt bridges between S42, R221, and D24 do not mediate allosteric communication in hGS, these interactions have a dramatic impact on the activity and structural stability of the enzyme.     

Interaction of Escherichia coli inorganic pyrophosphatase active sites

Escherichia coli inorganic pyrophosphatase (PPase) is a hexamer of identical subunits. This work shows that trimeric form of PPase exhibits the interaction of the active sites in catalysis. Some trimer subunits demonstrate high substrate binding affinity typical for hexamer whereas the rest of subunits reveal more than 300-fold substrate affinity decrease. This fact indicates the appearance of negative cooperativity of trimer subunits upon substrate binding. Association of the wild-type (WT) trimer with catalytically inactive, but still substrate binding mutant trimer into hexameric chimera restores the high activity of the first trimer, characteristic of trimer incorporated in the hexamer of WT PPase. Interaction of PPase active sites suggests that there are pathways for information transmission between the active sites, providing the perfect organization and concerted functioning of the hexameric active sites in catalysis.     

Comparison of ATP-pyrophosphate exchange reactions, catalyzed by two different "one-site" forms of tryptophanyl-tRNA-synthetase and by native enzyme

Kinetic parameters of the reaction of ATP-PPi exchange, catalysed by "one-site" form of beef pancreas tryptophanyl-tRNA-synthetase are determined. The one-site form was obtained by affinity labelling of one active centre of the enzyme with alkylated analog of tryptophan. The comparison of kinetic parameters of this one-site form with the other one produced earlier by affinity labelling with analog of tryptophanyl-tRNA and the native enzyme was carried out. It was shown that the presence of tRNA improves the discrimination properties of the enzyme and diminishes the concentrations of "dead-end" complexes. Negative cooperativity in binding of two molecules of tryptophan is observed, which disappears in the presence of ATP, leading to an apparent synergistic effect. The molecule of tryptophan, which is present in the active centre, diminishes affinity of pyrophosphate.     

Na+,K+-ATPase: structure, mechanism, and regulation

Structural organization of alpha- and beta-subunits of Na+,K+-ATPase in the membrane, the enzyme oligomeric structure, and mechanisms of ATP hydrolysis and cation transport are considered. The data on the structure of cation-binding sites and ion-conductive pathways of the pump are reviewed. The properties of isoforms of both subunits are described. Special attention was paid to the ATP modifying effect on Na+,K+-ATPase. To explain the rather complex dependence of the Na+,K+-ATPase activity on ATP concentration, a hypothesis is proposed, which is based on the assumption that the membrane contains the enzyme protomer exhibiting high affinity to ATP and an oligomer having low affinity to the nucleotide and characterized by positive cooperative interactions between subunits. Data on the Na+,K+-ATPase phosphorylation by protein kinases A and C are reviewed.     

Studies of the process of renaturation and assembly of Escherichia coli succinyl-CoA synthetase from its alpha and beta subunits

Succinyl-CoA synthetase catalyzes the substrate-level phosphorylation step of the tricarboxylic acid cycle. The enzyme, as isolated from Escherichia coli, has an alpha 2 beta 2 subunit structure. It is known that substrate-binding sites are distributed between both subunit types and that the active enzyme is the nondissociating tetramer. This paper describes a study of the process of assembly of the enzyme from its denatured constituent subunits. Starting with equimolar mixtures of the subunits that are prepared in denaturing conditions (6 M urea, 5% acetic acid), rapid renaturation to produce virtually a fully active enzyme occurs after neutralization and dilution under suitable conditions. This process occurs most efficiently in the presence of either ATP or Pi, indicating that occupation of the phosphoryl-binding site on the refolding alpha subunit facilitates productive intrasubunit interactions. We have determined conditions of protein concentration, pH, temperature, final urea concentration, and buffer compositions that optimize both the rate and extent of production of active enzyme. The final refolded product is indistinguishable from the native species with respect to its specific catalytic activity, size, and other physical properties. To probe further the mechanism and route of renaturation, we have shown that the rate of appearance of activity has first-order dependence on each of the two subunits. The step that determines the rate of assembly is thus bimolecular, such as the association of structural monomers to form a dimeric transient species. The highly specific mutual interactions between the refolding transient species of subunits must be essential for the correct assembly of this enzyme from the two gene products in vivo.     

On the role of substrate and GTP in the regulation of argininosuccinase activity.

As determined by equilibrium dialysis, bovine liver argininosuccinase of molecular weight 202,000 binds 4 mol of argininosuccinate or arginine/mol of enzyme. Negative homotropic interactions occur in the binding of both ligands at 0.15 ionic strength in the presence of phosphate. Argininosuccinate binds to two sites (Kdiss 1.6 times 10(-5) M) and four sites (Kdiss 2.9 times 10(-4) M) at low and high substrate concentration. Similarly, arginine binds to two sites (Kdiss 4.9 times 10(-4) M), and four sites (Kdiss 1.6 times 10(-3) M). At 0.05 ionic strength in Tris-HCl buffer, the four enzyme sites bind argininosuccinate independently and arginine binding remains negatively cooperative. Kinetic analysis gave double reciprocal plots that showed negative cooperatively also. The changes in Km were analogous to changes in Kdiss, thus indicating that the substrate binding sites correspond to catalytic sites. Since the catalytically active enzyme is a tetramer composed of four identical or closely similar subunits (Lusty, C.J., and Ratner, S. (1972) J. Biol. Chem. 247, 7010-7022), the present results show that each subunit contains one catalytic site. Ionic strength, phosphate ions, and GTP have each been found to influence negative cooperatively through a change in the affinity for argininosuccinate. The significance of the negative homotropic interactions and of the specific stimulation of activity by GTP is discussed with respect to different conformational forms of the enzyme and the in vivo regulation of argininosuccinase activity.     

The role of dissimilar subunits of NAD-specific isocitrate dehydrogenase from pig heart. Evaluation using affinity labeling

NAD-specific isocitrate dehydrogenase from pig heart is composed of three dissimilar subunits present in the native enzyme as 2 alpha:1 beta: 1 gamma, with a tetramer being the smallest form of complete enzyme. The role of these subunits has been explored using affinity labeling. Specifically labeled subunits are separated and then recombined with unmodified subunits to form dimers. Recombination of beta or gamma subunits modified by the isocitrate analogues, 3-bromo-2-ketoglutarate and 3,4-didehydro-2-ketoglutarate, with unmodified alpha subunit led to the same activity in the dimer as when unmodified beta or gamma was combined with alpha. Contrastingly, modification of alpha with these isocitrate analogues led to loss in activity either alone or when recombined with beta or gamma. Hence, the isocitrate site on alpha is required for catalytic activity but the isocitrate sites on beta or gamma are not necessary for the activity of the functional dimer. Reaction of isolated subunits with 3-bromo-2-ketoglutarate shows that alpha and the alpha beta dimer are modified at about the same rate as holoenzyme, suggestive of similarity of the isocitrate site in native enzyme and in isolated active entities containing alpha subunit; in contrast, beta and gamma subunits react more slowly. Modification by the 2',3'-dialdehyde derivative of the allosteric effector, ADP, led to loss of activity in reconstituted dimers, independent of which subunit was modified. Reaction of isolated subunits with the dialdehyde derivative of ADP is slow compared to the initial reaction with native enzyme, indicating differences in the effects of ADP on intact enzyme and subunits. The ADP sites on all subunits may thus be important in intersubunit interactions, which in turn modulate catalytic activity.     

Properties of chloroplast F1-ATPase partially modified by 2-azido adenine nucleotides, including demonstration of three catalytic pathways

Previous investigations on the distribution of [18O]Pi isotopomers formed by hydrolysis of [gamma-18O]ATP by the chloroplast F1-ATPase (CF1) showed that a single reaction pathway is used by all participating sites and that the pathway is modulated by ATP concentration as expected for cooperative interactions between catalytic sites. Such oxygen exchange measurements have been applied to CF1 modified at a single catalytic or noncatalytic site by 2-azido adenine nucleotides. When less than one catalytic or one noncatalytic site per enzyme is modified, hydrolysis occurs in part by the pathway of the unmodified enzyme plus at least one additional pathway at 200 microM and two additional pathways at 4 microM [gamma-18O]ATP. Thus, three sites are potentially catalytically active. The two new pathways shown by the derivatized enzyme logically can arise from nonidentical interactions of the remaining two underivatized beta subunits with the derivatized beta subunit. Reversals of bound ATP cleavage before Pi is released are increased, and the amount of product formed by the new pathways is changed when the ATP concentration is lowered. These modulations must result from the behavior of two remaining active catalytic sites rather than of one catalytic and one regulatory site. When the CF1 is derivatized more extensively, the original catalytic pathway is lost, and two catalytic pathways that do not show modulation by ATP concentration are found. The remaining beta subunits now have weak but independent catalytic capacity. In addition, the enzyme is no longer activated by Ca2+, loses MgGTPase activity, and is much less sensitive to azide.     

Studies of the functional topography of Escherichia coli RNA polymerase. Affinity labelling of RNA polymerase in a promoter complex by phosphorylating derivatives of primer oligonucleotides

Amidation of the 5'-phosphate group of the heptanucleotide pdApdApdApdTpdCpdGprC and of its derivatives of the general formula (pdN)npdGprC (n = 0-5) with imidazole, N-methylimidazole, and 4-dimethylaminopyridine afforded a series of phosphorylating affinity reagents. The parent oligonucleotides of this series complementary to promoter A2 of T7 phage over the region (-5 to +2) are known to be efficient primers of the synthesis of RNA by Escherichia coli RNA polymerase with promoter A2 as template. Treatment of the complex RNA-polymerase X promoter-A2 with affinity reagents followed by addition of [alpha-32P]UTP resulted in labelling of RNA polymerase by the residues -(pdN)npdGprCprU (p = radioactive phosphate). This affinity labelling was highly selective because elongation of the covalently bound residues (pdN)npdGprC by prU residues was catalyzed by the active center of RNA polymerase. The most efficient reagents were N-methylimidazolides. A dramatic change of the pattern of labelling of the subunits beta, beta', and sigma took place with changing n. Maximum labelling of the beta subunit occurred at n = 1 and of the sigma subunit at n = 5. The targets in both the subunits were His residues. The alpha subunit was not specifically labelled.     

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.     

Relation between Mg2+ activation and AMP inhibition of fructose-1,6-diphosphatase from rabbit skeletal muscles

It was shown that AMP, an allosteric inhibitor of fructose-1.6-bisphosphatase, decreases the apparent affinity of the enzyme for the activating cation, Mg2+, which is accompanied by a decrease of the kinetic cooperativity between the Mg2+-binding sites. In its turn, the Mg2+ increase diminishes the enzyme sensitivity to the inhibiting effect of AMP and decreases the cooperativity of the inhibitor binding. The heterotropic interactions between the allosteric inhibitor and activator binding centers are consistent with the predictions of the Monod-Wyman-Changeux model which involves two conformational states of the enzyme (of which one is catalytically inactive) differing in their affinity for the ligands. An increase in pH from 7.4 to 9.0 increases the enzyme affinity for Mg2+ and causes an equilibrium shift towards the catalytically active state of the enzyme.     

Studies of coenzyme binding to rabbit muscle glyceraldehyde-3-phoshate dehydrogenase.

The binding of NAD+, NADH and adenosine diphosphoribose (Ado-PP-Rib) to a stable, highly active and nucleotide-free preparation of rabbit muscle glyceraldehyde-3-phosphate dehydrogenase (D-glyceraldehyde-3-phosphate: NAD+ oxidoreductase (phosphorylating), EC 1.2.1.12) has been studied. All three nucleotides quench the protein fluorescence to the same extent when they bind to the enzyme, and this property has been used to measure the dissociation constants for the two high-affinity binding sites for the nucleotides. The results indicate negative interactions between, or non-identify of, these two binding sites, to which NAD+ and NADH bind with similar affinity. The binding of NAD+ to the enzyme has been studied by spectrophotometric titrations at 360 nm. It appears that the binding of NAD+ to each of the four subunits of the enzyme contributes equally to the intensity of this 'Racker' band. The dissociation constants associated with the binding of the third and fourth molecules of NAD+ estimated from such titrations confirm some previous estimates. The binding of NADH to the enzyme causes a decrease of intensity of the absorbance of the coenzyme at 340 nm, and the dissociation constants for binding of the third and fourth molecules of NADH have been estimated from spectrophotometric titrations. They are the same as those for NAD+. Judging by the apparent dissociation constants, negative interactions on binding the third molecule of NAD+ or NADH are more marked than those associated with the binding of the second and fourth molecules, suggesting that a major conformational change occurs at half-saturation of the tetramer with coenzyme.     

Phenylalanyl-tRNA synthetase from E. coli MRE-600: analysis of the active site distribution on the enzyme subunits by affinity labelling

Affinity labelling has been employed to localize the substrate-binding sites on the enzyme subunits of phenylalanyl-tRNA synthetase (L-phenylalanine:tRNAPhe-ligase, EC 6.1.1.20) of Escherichia coli MRE-600 (alpha 2 beta 2-type). N-Chlorambucilylphenylalanyl-tRNA, N-bromoacetylphenylalanyl-tRNA, tRNAPhe containing an azido group at the eighth position of the molecule (S4U), tRNAPhe containing azido groups at different points of the molecule, p-azidoanilidate of phenylalanine, adenosine 5'-trimethaphosphate and N-bromoacetyl-L-phenylalaninyladenylate were used in experiments. It has been shown that tRNA-binding sites are formed on heavy beta-subunits of the enzyme. Phenylalanyl-tRNA is also localized on beta-subunits, while the aminoacyl moiety of aminoacyl-tRNA is localized near the contact region of subunits. The phenylalanine-binding site is located on light alpha-subunits of the enzyme. Adenosine 5'-trimethaphosphate and the analogue of phenylalanyladenylate modify both types of enzyme subunits. In our opinion, the catalytic center of tRNA aminoacylation is formed in the contact region of subunits, and the aminoacyl moiety is transferred into tRNA (from the alpha- into beta-subunit in the region of their contact).     

Effect of subunit interactions on enzymatic activity of glutathione S-transferases: a radiation inactivation study.

The glutathione S-transferases are a family of dimeric enzymes. Three isozymes from the alpha family, termed YaYa, YaYc, and YcYc, and three from the mu family, termed Yb1Yb1, Yb1Yb2, and Yb2Yb2, were purified from rat liver. Binding studies were performed by equilibrium dialysis using a radiolabeled product, S(-)[14C](dinitrophenyl)glutathione. Each isozyme contained two independent binding sites which had equal affinity for the ligand. The presence of two independent active sites per enzyme dimer suggests that each subunit contains a complete active site. This conclusion was examined further using radiation inactivation which also allowed for assessment of the importance of subunit interactions in catalytic activity. The activity target size of YaYa (47 kDa) was significantly larger than the protein monomer target size (31 kDa); similarly the activity target size of YaYc was that of the dimer (54 kDa). In contrast, the activity target sizes of Yb1Yb1 and Yb2Yb2 were the same, being 35 and 29 kDa, respectively, and the protein monomer target size of Yb1Yb1 also was similar, being 32 kDa. These data indicate that interactions between subunits are critical for the maintenance of enzymatic activity of alpha class enzymes whereas each subunit of the two mu class proteins is capable of independent catalytic activity.     

The binding of oxidized coenzymes by glutamate dehydrogenase and the effects of glutarate and purine nucleotides

1. The binding of NAD(+) and NADP(+) to glutamate dehydrogenase has been studied in sodium phosphate buffer, pH7.0, by equilibrium dialysis. Approximate values for the dissociation constants are 0.47 and 2.5mm respectively. For NAD(+) the value agrees with that estimated from initial-rate results. 2. In the presence of the substrate analogue glutarate both coenzymes are bound more firmly, and there is one active centre per enzyme subunit. The binding results cannot be described in terms of independent and identical active centres, and binding is stronger at low coenzyme concentrations than at high concentrations. Either the six subunits of the oligomer are not identical or there are negative interactions between them in the binding of coenzymes in ternary complexes with glutarate. The latter explanation is favoured. 3. The binding studies support the conclusions drawn from earlier kinetic studies of the glutamate reaction. 4. ADP and GTP respectively decrease and increase the affinity of the enzyme for NAD(+) and NADP(+), in both the presence and absence of glutarate. The negative binding interactions in the presence of glutarate are abolished by ADP, which decreases the affinity for the coenzymes at low concentrations of the latter. 5. In the presence of glutarate, GTP and NAD(+) or NADP(+), the association of enzyme oligomers is prevented, and the solubility of the enzyme is decreased; the complex of enzyme and ligands readily crystallizes. 6. The results are discussed in relation to earlier kinetic studies.     

Regulation of mitochondrial malate dehydrogenase: kinetic modulation independent of subunit interaction

Porcine heart mitochondrial malate dehydrogenase (EC 1.1.1.37), a dimeric enzyme of Mr = 70,000, is both allosterically activated and inhibited by citrate. Using an affinity elution procedure based upon citrate binding to malate dehydrogenase, the isolation of pure heterodimer (a dimeric species with one active subunit and one iodoacetamide-inactivated subunit) has been achieved. Investigations utilizing this heterodimer in conjunction with resin-bound monomers of malate dehydrogenase have allowed the formulation of a definite conclusion concerning the role of subunit interactions in catalysis and regulation of this enzyme. The citrate kinetic effects, oxaloacetate inhibition, malate activation, and the effects of 2-thenoyl-trifluoroacetone (TTFA) are shown to be independent of interaction between catalytically active subunits. Previous kinetic data thought to support a reciprocating catalytic mechanism for this enzyme may be reinterpreted upon closer analysis in relation to an allosteric, conformationally specific binding model for malate dehydrogenase.     

Tyrosyl-tRNA synthetase acts as an asymmetric dimer in charging tRNA. A rationale for half-of-the-sites activity

Tyrosyl-tRNA synthetase from Bacillus stearothermophilus is a classical example of an enzyme with half-of-the-sites activity. The enzyme crystallizes as a symmetrical dimer that is composed of identical subunits, each having a complete active site. In solution, however, tyrosyl-tRNA synthetase binds tightly, and activates rapidly, only 1 mol of Tyr/mol of dimer. It has recently been shown that the half-of-the-sites activity results from an inherent asymmetry of the enzyme. Only one subunit catalyzes formation of Tyr-AMP, and interchange of activity between subunits is not detectable over a long time scale. Paradoxically, however, the kinetics of tRNA charging are biphasic with respect to [Tyr], suggesting that both subunits of the dimer are catalytically active. This paradox has now been resolved by kinetic analysis of heterodimeric enzymes containing different mutations in each subunit. Biphasic kinetics with unchanged values of KM for Tyr are maintained when one of the two tRNA-binding domains is removed and also when the affinity of the "inactive" site for Try is reduced by 2-58-fold. The biphasic kinetics do not result from catalysis at both active sites, but instead appear to result from two molecules of Tyr binding sequentially to the same site. A second molecule of Tyr perhaps aids the dissociation of Tyr-tRNA by displacing the tyrosyl moiety from its binding site. A monomer of the enzyme is probably too small to allow both recognition and aminoacylation of a tRNA molecule. This could explain the requirement for the enzyme to function as an asymmetric dimer.