Yeast epiarginase regulation,an enzyme-enzyme activity control: identification of residues of ornithine carbamoyltransferase and arginase responsible for enzyme catalytic and regulatory activities

In the presence of ornithine and arginine, ornithine carbamoyltransferase (OTCase) and arginase form a one-to-one enzyme complex in which the activity of OTCase is inhibited whereas arginase remains catalytically active. The mechanism by which these nonallosteric enzymes form a stable complex triggered by the binding of their respective substrates raises the question of how such a cooperative association is induced. Analyses of mutations in both enzymes identify residues that are required for their association, some of them being important for catalysis. In arginase, two cysteines at the C terminus of the protein are crucial for its epiarginase function but not for its catalytic activity and trimeric structure. In OTCase, mutations of putative ornithine binding residues, Asp-182, Asn-184, Asn-185, Cys-289, and Glu-256 greatly reduced the affinity for ornithine and impaired the interaction with arginase. The four lysine residues located in the SMG loop, Lys-260, Lys-263, Lys-265, and Lys-268, also play an important role in mediating the sensitivity of OTCase to ornithine and to arginase and appear to be involved in transducing and enhancing the signal given by ornithine for the closure of the catalytic domain.     

Metabolic enzymes from psychrophilic bacteria: challenge of adaptation to low temperatures in ornithine carbamoyltransferase from Moritella abyssi

The enzyme ornithine carbamoyltransferase (OTCase) of Moritella abyssi (OTCase(Mab)), a new, strictly psychrophilic and piezophilic bacterial species, was purified. OTCase(Mab) displays maximal activity at rather low temperatures (23 to 25 degrees C) compared to other cold-active enzymes and is much less thermoresistant than its homologues from Escherichia coli or thermophilic procaryotes. In vitro the enzyme is in equilibrium between a trimeric state and a dodecameric, more stable state. The melting point and denaturation enthalpy changes for the two forms are considerably lower than the corresponding values for the dodecameric Pyrococcus furiosus OTCase and for a thermolabile trimeric mutant thereof. OTCase(Mab) displays higher K(m) values for ornithine and carbamoyl phosphate than mesophilic and thermophilic OTCases and is only weakly inhibited by the bisubstrate analogue delta-N-phosphonoacetyl-L-ornithine (PALO). OTCase(Mab) differs from other, nonpsychrophilic OTCases by substitutions in the most conserved motifs, which probably contribute to the comparatively high K(m) values and the lower sensitivity to PALO. The K(m) for ornithine, however, is substantially lower at low temperatures. A survey of the catalytic efficiencies (k(cat)/K(m)) of OTCases adapted to different temperatures showed that OTCase(Mab) activity remains suboptimal at low temperature despite the 4.5-fold decrease in the K(m) value for ornithine observed when the temperature is brought from 20 to 5 degrees C. OTCase(Mab) adaptation to cold indicates a trade-off between affinity and catalytic velocity, suggesting that optimization of key metabolic enzymes at low temperatures may be constrained by natural limits.     

Crystal structure of human ornithine transcarbamylase complexed with carbamoyl phosphate and L-norvaline at 1.9 A resolution

The crystal structure of human ornithine transcarbamylase (OTCase) complexed with carbamoyl phosphate (CP) and L-norvaline (NOR) has been determined to 1.9-A resolution. There are significant differences in the interactions of CP with the protein, compared with the interactions of the CP moiety of the bisubstrate analogue N-(phosphonoacetyl)-L-ornithine (PALO). The carbonyl plane of CP rotates about 60 degrees compared with the equivalent plane in PALO complexed with OTCase. This positions the side chain of NOR optimally to interact with the carbonyl carbon of CP. The mixed-anhydride oxygen of CP, which is analogous to the methylene group in PALO, interacts with the guanidinium group of Arg-92; the primary carbamoyl nitrogen interacts with the main-chain carbonyl oxygens of Cys-303 and Leu-304, the side chain carbonyl oxygen of Gln-171, and the side chain of Arg-330. The residues that interact with NOR are similar to the residues that interact with the ornithine (ORN) moiety of PALO. The side chain of NOR is well defined and close to the side chain of Cys-303 with the side chains of Leu-163, Leu-200, Met-268, and Pro-305 forming a hydrophobic wall. C-delta of NOR is close to the carbonyl oxygen of Leu-304 (3.56 A), S-gamma atom of Cys-303 (4.19 A), and carbonyl carbon of CP (3.28 A). Even though the N-epsilon atom of ornithine is absent in this structure, the side chain of NOR is positioned to enable the N-epsilon of ornithine to donate a hydrogen to the S-gamma atom of Cys-303 along the reaction pathway. Binding of CP and NOR promotes domain closure to the same degree as PALO, and the active site structure of CP-NOR-enzyme complex is similar to that of the PALO-enzyme complex. The structures of the active sites in the complexes of aspartate transcarbamylase (ATCase) with various substrates or inhibitors are similar to this OTCase structure, consistent with their common evolutionary origin.     

Regulation of arginine metabolism in Saccharomyces cerevisiae. Association of arginase and ornithine transcarbamoylase

Association of arginase and ornithine transcarbamoylase (OTCase) has been proposed to play an essential role in the regulation of arginine metabolism in Saccharomyces cerevisiae (Wiame, J.-M. (1971) Curr. Top. Cell. Reg. 4, 1-39). In this report multienzyme complex formation is directly demonstrated in the presence of the active-site ligands for OTCase and arginase. Using equilibrium sedimentation, a dissociation constant for complex formation was determined to be 2.3 X 10(-8) M in the presence of ornithine and agmatine, active-site ligands for OTCase and arginase, respectively. A molecular stoichiometry in the complex of one molecule of OTCase to one molecule of arginase was verified using transmission electron microscopy. The dimensions of the complex were determined by negative staining and rotary and unidirectional shadowing techniques to be 102 A wide by 81 A high. These dimensions are quantitively consistent with dimensions of the individual enzymes (Duong, L. T., Eisenstein, E., Green, S. M., Ornberg, R. L., and Hensley, P. (1986) J. Biol. Chem. 261, 12807-12813). The enzymatic activity of OTCase is virtually completely inhibited when associated with arginase, reflecting the dramatic modulation of enzyme activity as a consequence of the acquisition of quaternary structure in this multienzyme complex.     

Crystal structures of Bacillus caldovelox arginase in complex with substrate and inhibitors reveal new insights into activation, inhibition and catalysis in the arginase superfamily

BACKGROUND: Arginase is a manganese-dependent enzyme that catalyzes the hydrolysis of L-arginine to L-ornithine and urea. In ureotelic animals arginase is the final enzyme of the urea cycle, but in many species it has a wider role controlling the use of arginine for other metabolic purposes, including the production of creatine, polyamines, proline and nitric oxide. Arginase activity is regulated by various small molecules, including the product L-ornithine. The aim of these structural studies was to test aspects of the catalytic mechanism and to investigate the structural basis of arginase inhibition. RESULTS: We report here the crystal structures of arginase from Bacillus caldovelox at pH 5.6 and pH 8.5, and of binary complexes of the enzyme with L-arginine, L-ornithine and L-lysine at pH 8.5. The arginase monomer comprises a single compact alpha/beta domain that further associates into a hexameric quaternary structure. The binary complexes reveal a common mode of ligand binding, which places the substrate adjacent to the dimanganese centre. We also observe a conformational change that impacts on the active site and is coupled with the occupancy of an external site by guanidine or arginine. CONCLUSIONS: The structures reported here clarify aspects of the active site and indicate key features of the catalytic mechanism, including substrate coordination to one of the manganese ions and an orientational role for a neighboring histidine residue. Stereospecificity for L-amino acids is found to depend on their precise recognition at the active-site rim. Identification of a second arginine-binding site, remote from the active site, and associated conformational changes lead us to propose a regulatory role for this site in substrate hydrolysis.     

Studies of ligand binding to Escherichia coli adenylosuccinate synthetase

Dissociation constants of Escherichia coli adenylosuccinate synthetase with IMP, GTP, adenylosuccinate, and AMP (a competitive inhibitor for IMP) were determined by measuring the extent of quenching of the intrinsic tryptophan fluorescence of the enzyme. The enzyme has one binding site for each of these ligands. Aspartate and GDP did not quench the fluorescence to any great extent, and their dissociation constants could not be determined. These ligand binding studies were generally supportive of the kinetic mechanism proposed earlier for the enzyme. Cys291 was modified with the fluorescent chromophores N-(iodoacetylaminoethyl)-5-naphthylamine-1-sulfonate and tetramethylrhodamine maleimide in order to measure enzyme conformational changes attending ligand binding. The excitation and emission spectra of these fluorophores are not altered by the addition of active site binding ligands. TbGTP and TbGDP were used as native reporter groups, and changes in their fluorescence on complexing with the enzyme and various ligands made it possible to detect conformational changes occurring at the active site. Evidence is presented for abortive complexes of the type: enzyme-TbGTP-adenylosuccinate and enzyme-TbGTP-adenylosuccinate-aspartate. These results suggest that the IMP and aspartate binding sites are spatially separated.     

Binding of allosteric effectors to carbamyl-phosphate synthetase from Escherichia coli.

The binding of ornithine and inosine 5'-monophosphate (IMP), positive allosteric effectors, and of uridine 5'-monophosphate (UMP), a negative allosteric effector, to carbamyl-phosphate synthetase from Escherichia coli was studied by the technique of equilibrium dialysis. The monomeric form of the enzyme has one binding site for each of the three allosteric ligands. The binding of UMP is inhibited by ornithine, IMP, MgATP, and ammonia (also a positive allosteric effector). Bicarbonate, L-glutamine, and adenosine 5'-triphosphate (ATP) (Mg2+ absent) had no effect on the binding of UMP. The affinity of the enzyme for UMP was increased if phosphate buffer was replaced by 2-amino-2-hydroxymethyl-1,3-propanediol (Tris) buffer. The binding of ornithine was inhibited by UMP and ammonia, enhanced by MgATP, MgADP, and IMP, and not affected by bicarbonate, L-glutamine, or ATP (Mg2+ absent). Ornithine and ammonia probably bind to the same site on the enzyme. The binding of IMP is facilitated by ornithine and ammonia, but is inhibited by MgATP or ATP, indicating that adenine nucleotides can also bind to the IMP binding site. The results of these binding studies are consistent with a scheme previously proposed in which the allosteric effectors function by stabilizing one or the other of two different conformational states of the enzyme which are in equilibrium with each other (Anderson, P.M., and Marvin, S.V. (1970), Biochemistry 9, 171). According to this scheme, binding of the substrate MgATP is greatly facilitated when the enzyme exists in the conformational state stabilized by the positive allosteric effectors.     

Quantitative relationships of site to site interaction in Escherichia coli D-3-phosphoglycerate dehydrogenase revealed by asymmetric hybrid tetramers

A set of asymmetric hybrid tetramers of Escherichia coli d-3-phosphoglycerate dehydrogenase (PGDH) have been made by gene co-expression and KSCN-induced dimer exchange. These tetramers contain varied numbers of active sites and effector binding sites arranged in different orientations within the tetramer. They reveal that PGDH displays half-of-the-sites activity with respect to its active sites and that the two sites that are active at any particular time lie in subunits on either side of the nucleotide binding domain interface. Half-of-the-sites functionality is also observed for the effector even though all four sites eventually bind effector. That is, only two effector sites need to be occupied for maximum inhibition. Binding of the last two effector molecules does not contribute functionally to inhibition of activity. Furthermore, positive cooperativity of inhibition of activity by the effector is completely dependent on the positive cooperativity of binding of the effector. Binding of the first effector molecule produces a conformational change that essentially completely inhibits the active site within the subunit to which it binds and produces an approximate 33% inhibition of the active site in the subunit to which it is not bound. Binding of the second effector at the opposite regulatory domain interface completes the inhibition of activity. This simple relationship defines the positional and quantitative influence of effector ligand binding on activity and can be used to predict the maximum level of inhibition of individual hybrid tetramers. In addition, the site-specific quantitative relationship of effector binding to individual active sites can be used to model the inhibition profile with relatively good agreement. These simple rules for the site to site interaction in PGDH provide significant new insight into the mechanism of allostery of this enzyme.     

Structural similarity between ornithine and aspartate transcarbamoylases of Escherichia coli: characterization of the active site and evidence for an interdomain carboxy-terminal helix in ornithine transcarbamoylase.

Predictions of tertiary structures of proteins from their amino acid sequences are facilitated greatly when the structures of homologous proteins are known. On this basis, structural features of Escherichia coli ornithine transcarbamoylase (OTCase) were investigated by site-directed mutagenesis experiments based on the known tertiary structure of the catalytic (c) chain of E. coli aspartate transcarbamoylase (ATCase). In ATCase, each c chain is composed of two globular domains connected by two interdomain helices, one of which is near the C-terminus and is critical for the in vivo folding of the chains and their assembly into trimers. Each active site is located at the interface between two chains and requires the participation of residues from each of the adjacent chains. OTCase, a trimeric enzyme, has been proposed to be similar in structure to the ATCase trimer on the basis of sequence identity (32%), the nature of the reaction catalyzed by the enzyme, and secondary structure predictions. As shown here, analysis of OTCase and ATCase sequences revealed extensive evolutionary conservation in portions corresponding to the ATCase active site and the C-terminal helix. Truncations and substitutions within the predicted C-terminal helix of OTCase had effects on activity and thermal stability strikingly similar to those caused by analogous alterations in ATCase. Similarly, substitutions at either of two conserved residues, Ser 55 and Lys 86, in the proposed active site of OTCase had deleterious effects parallel to those caused by the analogous ATCase substitutions. Hybrid trimers comprised of chains from both these relatively inactive OTCase mutants exhibited dramatically increased activity, as predicted for shared active sites located at the chain interfaces. These results strongly support the hypothesis that the tertiary and quaternary structures of the two enzymes are similar.     

Converting catabolic ornithine carbamoyltransferase to an anabolic enzyme

Pseudomonas aeruginosa has an anabolic and a catabolic ornithine carbamoyltransferase (OTCase). In vitro, these homologous enzymes catalyze the same reaction (ornithine + carbamoyl phosphate (CP) in equilibrium citrulline + Pi), yet in vivo they function unidirectionally owing to specific kinetic properties. The catabolic OTC-ase cannot promote the anabolic reaction (citrulline formation) in vivo because of a sigmoidal CP saturation curve and a high CP concentration for half-maximal velocity. The structural basis for this kinetic specialization was examined. The catabolic OTCase lost most of its homotropic cooperativity and gained anabolic activity when an amino acid residue near the CP binding site, Glu-106, was replaced by alanine or glycine. In the anabolic OTCase of Escherichia coli the glutamine residue corresponding to Glu-106 was exchanged for glutamate; however, in this case no CP cooperativity was acquired. Thus, in catabolic OTCase, sequence features in addition to Glu-106 are important for sigmoidal CP saturation, and such a sequence was identified in the C-terminal part. By an in vivo gene fusion technique the 9 C-terminal amino acids of catabolic OTCase were replaced by the homologous 8 amino acids from anabolic OTCase of E. coli; the hybrid enzyme had a markedly reduced homotropic cooperativity. This gene fusion method should be generally useful for directed enzyme evolution.     

Effects of replacement of active site residue glutamine 231 on activity and allosteric properties of aspartate transcarbamoylase.

Since crystallographic studies on Escherichia coli aspartate transcarbamoylase (ATCase) indicate that Gln 231 is in the active site of the enzyme and participates in the binding of the substrate, aspartate, it seemed of interest to examine mutant enzymes in which Gln 231 was replaced by Asn or Ile. The two mutant forms containing amino acid substitutions were characterized by a combination of steady-state kinetics, hydrodynamic measurements, and equilibrium ligand binding techniques. Both mutant forms exhibited a dramatic reduction in the affinity of the protein for substrates and substrate analogues as well as a very large decrease in catalytic activity. Moreover, the amino acid substitutions introduced within the active site of the enzyme led to unusual allosteric properties in the mutant enzymes. Although the bisubstrate analogue N-(phosphonoacetyl)-L-aspartate promotes the characteristic global conformational change in the mutant forms that is observed with the wild-type enzyme, the combination of substrate and substrate analogue does not. Cooperativity with respect to substrate binding is largely reduced compared to wild-type ATCase. Also, the effector molecules ATP and CTP which bind to the regulatory chains have dramatic effects on the activity of the mutant enzymes containing replacements for Gln 231 in the catalytic chains. In stark contrast to the wild-type enzyme, in which effects of nucleotides are manifested primarily by changes in the K0.5 of the enzyme, ATP and CTP have large effects on the Vmax of the mutant enzymes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Stabilization of the shikimate pathway enzyme dehydroquinase by covalently bound ligand

Reversible binding of a ligand to an enzyme active site can elicit a variety of changes in the protein, such as conformational changes (close to the site of binding or communicated over long distances), changes in the ionization state of surrounding amino acid side chains, changes in the interaction of the target protein with other subunits (or other proteins), or even changes in the thermodynamic stability of the protein. Relatively little attention has been given to studying these effects in proteins to which the ligand has been irreversibly bound, yet this can be a convenient way of studying the effects of ligand binding in the absence of association/dissociation equilibria. We report the dramatic changes which occur to the shikimate pathway enzyme dehydroquinase when ligand is attached to its active site after borohydride reduction of the mechanistically important Schiff's base intermediates. The effects of this modification have been characterized by limited proteolysis, circular dichroism, guanidine hydrochloride denaturation, and differential scanning calorimetry. The conclusions from these studies are that although anchoring the ligand at the active site does not cause a gross change in conformation, it does increase markedly the conformational stability of the protein. This is conclusively established by three separate experiments: 1) the modified protein is completely resistant to proteases, whereas the unmodified protein is very susceptible to proteolysis; 2) the concentration of guanidine hydrochloride required to unfold the ligand-linked dehydroquinase is 3-4-fold greater than that of the unmodified protein; 3) the melting temperature (Tm) of the modified protein is 40 degrees C higher than that of the unmodified protein. These results are a very clear example of the thermodynamic link between ligand binding, conformational stability, and proteolytic susceptibility in vitro and will be a useful system for dissecting the contributions of individual protein-ligand interactions to these parameters.     

Effects of active site modification and reversible dissociation on the secondary structure of triosephosphate isomerase.

Binding of ligands to the catalytic center of mammalian triosephosphate isomerase (TPI) induces a conformational change(s) that enhances the specific deamidation of Asn71 at the subunit interface. Deamidation initiates dissociation and degradation of the enzyme in vivo and in vitro. We have utilized circular dichroism spectroscopy to examine the conformational changes in the enzyme upon ligand binding and subunit dissociation/reassociation. Native TPI from rabbit, chicken, and yeast exhibit similar spectra at pH 7.5, but are substantially different at pH 9.5. Covalent reaction of the active site Glu 165 with the substrate analogue 3-chloroacetol phosphate results in a conformational change (decrease in beta-sheet) which is similar in TPI from all three species. Reversible dissociation of the dimeric enzyme in guanidine followed by dialysis, although permitting full recovery of catalytic activity, results in refolded dimers with decreased alpha-helix. These conformational changes induced by ligand binding, pH, or reversible dissociation explain, in part, the differences in the chemical and physical properties of the enzyme from the three species at alkaline pH, the increased lability of the dissociated/reassociated enzyme, and corroborate 31P NMR data on substrate-induced conformational changes. These studies also support the concept of molecular wear and tear whereby ligand binding at the catalytic center induces conformational changes that increase the probability of covalent modification and ultimate degradation of the protein.     

Inhibitors of different structure induce distinguishing conformations in the omega loop,Cys69-Cys96, of mouse acetylcholinesterase

We have shown previously that association of reversible active site ligands induces a conformational change in an omega loop (Omega loop), Cys(69)-Cys(96), of acetylcholinesterase. The fluorophore acrylodan, site-specifically incorporated at positions 76, 81, and 84, on the external portion of the loop not lining the active site gorge, shows changes in its fluorescence spectrum that reflect the fluorescent side chain moving from a hydrophobic environment to become more solvent-exposed. This appears to result from a movement of the Omega loop accompanying ligand binding. We show here that the loop is indeed flexible and responds to conformational changes induced by both active center and peripheral site inhibitors (gallamine and fasciculin). Moreover, phosphorylation and carbamoylation of the active center serine shows distinctive changes in acrylodan fluorescence spectra at the Omega loop sites, depending on the chirality and steric dimensions of the covalently conjugated ligand. Capping of the gorge with fasciculin, although it does not displace the bound ligand, dominates in inducing a conformational change in the loop. Hence, the ligand-induced conformational changes are distinctive and suggest multiple loop conformations accompany conjugation at the active center serine. The fluorescence changes induced by the modified enzyme may prove useful in the detection of organophosphates or exposure to cholinesterase inhibitors.     

Immobilization of arginase and its application in an enzymatic chromatographic column: thermodynamic studies of nor-NOHA/arginase binding and role of the reactive histidine residue

A biochromatographic approach is developed to measure for the first time changes in enthalpy, heat capacity change and protonation for the binding of nor-NOHA to arginase in a wide temperature range. For this, the arginase enzyme was immobilized on a chromatographic support. It was established that this novel arginase column was stable during an extended period of time. The affinity of nor-NOHA to arginase is high and changes slightly with the pH, because the number of protons linked to binding is low. The determination of the enthalpy change at different pH values suggested that the protonated group in the nor-NOHA-arginase complex exhibits a heat protonation of approximately -33 kJ/mol. This value agrees with the protonation of an imidazole group. Our result confirmed that active-site residue Hist 141 is protonated as imidazolium cation. Hist 141 can function as a general acid to protonate the leaving amino group of L-ornithine during catalysis. The thermodynamic data showed that nor-NOHA-arginase binding, for low temperature (<15 degrees C), is enthalpically unfavourable and being dominated by a positive entropy change. This result suggests that dehydration at the binding interface and charge-charge interactions contribute to the nor-NOHA-arginase complex formation. The temperature dependence of the free energy of binding is weak because of the enthalpy-entropy compensation caused by a large heat capacity change, DeltaC(p)=-2.43 kJ/mol/K, of arginase. Above 15 degrees C, the thermodynamic data DeltaH and DeltaS became negative due to van der Waals interactions and hydrogen bonding which are engaged at the complex interface confirming strong enzyme-inhibitor hydrogen bond networks. As well, by the use of these thermodynamic data and known correlations it was clearly demonstrated that the binding of nor-NOHA to arginase produces slight conformational changes in the vicinity of the active site. Our work indicated that our biochromatographic approach could soon become very attractive for studying other enzyme-ligand binding.     

Characterization of the conformational changes of acetohydroxy acid isomeroreductase induced by the binding of Mg2+ ions, NADPH, and a competitive inhibitor.

Acetohydroxy acid isomeroreductase (EC 1.1.1.86), the second enzyme of the parallel branched chain amino acid pathway, is a homodimer with an Mr of approximately 114000 which in the presence of Mg2+ ions catalyzes an unusual alkyl migration followed by an NADPH-dependent reduction. Prior binding of NADPH and Mg2+ to the enzyme was shown to be required for substrate or competitive inhibitor [N-hydroxy-N-isopropyloxamate (IpOHA)] binding [Dumas, R., et al. (1994) Biochem. J. 301, 813-820]. Moreover, crystallographic data for the enzyme-NADPH-Mg2+-IpOHA complex [Biou, V., et al. (1997) EMBO J. 16, 3405-3415] have shown that IpOHA was completely buried inside the active site. These observations raised the question of how the reaction intermediate analogue inhibitor can reach the active site and implied that conformational changes occurred during the binding process. With a view of characterizing these conformational changes, H-D exchange experiments combined with mass spectrometry were performed. Results demonstrated that Mg2+ ions and NADPH binding led to an initial conformational change at the interface of the two domains of each monomer. Binding of the two cofactors to isomeroreductase alters the structure of the active site to promote inhibitor (substrate) binding, in agreement with the ordered mechanism of the enzyme. Structural changes remote from the active site were also found. They were interpreted as long-range structural effects on the two domains and on the two monomers in the time course of the ligand binding process.     

Thermodynamic compensation upon binding to exosite 1 and the active site of thrombin

Several lines of experimental evidence including amide exchange and NMR suggest that ligands binding to thrombin cause reduced backbone dynamics. Binding of the covalent inhibitor dPhe-Pro-Arg chloromethyl ketone to the active site serine, as well as noncovalent binding of a fragment of the regulatory protein, thrombomodulin, to exosite 1 on the back side of the thrombin molecule both cause reduced dynamics. However, the reduced dynamics do not appear to be accompanied by significant conformational changes. In addition, binding of ligands to the active site does not change the affinity of thrombomodulin fragments binding to exosite 1; however, the thermodynamic coupling between exosite 1 and the active site has not been fully explored. We present isothermal titration calorimetry experiments that probe changes in enthalpy and entropy upon formation of binary ligand complexes. The approach relies on stringent thrombin preparation methods and on the use of dansyl-l-arginine-(3-methyl-1,5-pantanediyl)amide and a DNA aptamer as ligands with ideal thermodynamic signatures for binding to the active site and to exosite 1. Using this approach, the binding thermodynamic signatures of each ligand alone as well as the binding signatures of each ligand when the other binding site was occupied were measured. Different exosite 1 ligands with widely varied thermodynamic signatures cause a similar reduction in ΔH and a concomitantly lower entropy cost upon DAPA binding at the active site. The results suggest a general phenomenon of enthalpy-entropy compensation consistent with reduction of dynamics/increased folding of thrombin upon ligand binding to either the active site or exosite 1.     

Crystal structure of unliganded phosphofructokinase from Escherichia coli

In an attempt to characterize the mechanism of co-operativity in the allosteric enzyme phosphofructokinase from Escherichia coli, crystals were grown in the absence of activating ligands. The crystal structure was determined to a resolution of 2.4 A by the method of molecular replacement, using the known structure of the liganded active state as a starting model, and has been refined to a crystallographic R-factor of 0.168 for all data. Although the crystallization solution would be expected to contain the enzyme in its inactive conformation, with a low affinity for the co-operative substrate fructose 6-phosphate, the structure in these crystals does not show the change in quaternary structure seen in the inactive form of the Bacillus stearothermophilus enzyme (previously determined at low resolution), nor does it show any substantial change in the fructose 6-phosphate site from the structure seen in the liganded form. Compared to the liganded form, there are considerable changes around the allosteric effector site, including the disordering of the last 19 residues of the chain. It seems likely that the observed conformation corresponds an active unliganded form, in which the absence of ligand in the effector site induces structural changes that spread through much of the subunit, but cause only minor changes in the active site. It is not clear why the crystals should contain the enzyme in a high-affinity conformation, which presumably represents only a small fraction of the molecules in the crystallizing solution. However, this structure does identify the conformational changes involved in binding of the allosteric effectors.     

Modification of the regulatory properties of pyruvate kinase of Neurospora by growth at elevated temperatures.

Pyruvate kinase (EC 2.7.1.40) was isolated from Neurospora crassa mycelium grown at 28 degrees C (PK-28) and at 42 degrees C (PK-42). The regulatory properties, particularly the response towards the allosteric effector fructose 1,6-diphosphate (FDP), was different in the two enzymes. PK-28 showed an activation by FDP but PK-42, under comparable conditions, appeared to be activated by low concentrations of FDP and inhibited by higher ones. For PK-28, complex formation with FDP results in a lowering of the isoelectric point from 6.40 to 5.50, representing the pI of the unliganded enzyme and that of the complex, respectively. In contrast to this, PK-42 exhibits a weak binding to FDP as suggested by a lack of decrease in the isoelectric point on treatment with comparable concentrations of FDP. Studies with quenching of aromatic residue fluorescence of PK-28 and PK-42, following binding of FDP, indicate that although this ligand binds to both types of enzymes the affinity for the two is vastly different. Dissociation constants of 9.3 muM and 0.1 mM were calculated for the binding of FDP to PK-28 and PK-42, respectively. It is concluded that growth at elevated temperatures induced a conformational change in the pyruvate kinase leading to partial desensitization of the allosteric site. The nature of the factor(s) responsible for this change is not understood at present.