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Alpha 1 -proteinase inhibitor (A 1 PI) deficiency is a genetic condition predisposing to emphysema. Respreeza/Zemaira, a therapeutic preparation of A 1 PI, is prepared from human plasma. This article describes the purity and stability of Respreeza/Zemaira and the capacity of virus and prion reduction steps incorporated into its manufacturing process. Purity and stability of Respreeza/Zemaira were analysed using established methods. To test pathogen clearance capacity, high levels of test viruses/prions were spiked into aliquots of production intermediates and clearance studies were performed for selected manufacturing steps, under production and robustness conditions, using validated scale-down models. Respreeza/Zemaira had a purity of 99% A 1 PI and consisted of 96% monomers. It remained stable after storage for 3 years at 25 °C. Specific activity was 0.895 mg active A 1 PI/mg protein. Pasteurisation inactivated enveloped viruses and the non-enveloped hepatitis A virus. 20 N/20 N virus filtration was highly effective and robust at removing all tested viruses, including parvoviruses, to below the limit of detection. Cold ethanol fractionation provided substantial reduction of prions. The manufacturing process of Respreeza/Zemaira ensures the production of a stable and pure product. Taking into consideration the donor selection process, the testing of donations, and the highly effective virus and prion reduction, Respreeza/Zemaira has a high safety margin.

Geological storage of CO2 is receiving growing attention by industry as an option to reduce CO2 emissions. For this, it is indispensable to develop geochemical models for the interaction of CO2 and incidental components like SO2 with formation rocks and brines. To establish such whole-rock models it is essential to have a good understanding of the chemical reactions, thermodynamic properties, and kinetic data of mono-mineral systems. In the German project “CO2 Purity for Capture and Storage (COORAL)” several institutes are working on the requirements for the purity of the CO2 stream during capture, transport, injection, and storage. Geochemical modeling and laboratory experiments on water-rock-gas interactions are carried out to improve understanding of mineral alteration processes. In whole- rock models of a generic Buntsandstein (sandstone), short-term precipitation of anhydrite occurred when SO2 was present. Furthermore, increasing feldspar dissolution and related illitisation reactions for the system CO 2 -SO2 in comparison to pure CO2 are of importance.

Modelling fluid–rock interactions induced by CO2 is a key issue when evaluating the technical feasibility and long-term safety assessment of CO2 storage projects in deep formations. The German R&D programme CLEAN (CO2 Large-Scale Enhanced Gas Recovery in the Altmark Natural Gas Field) investigated the almost depleted onshore gas reservoir located in the Rotliegend sandstone at over 3,000-m depth. The high salinity of the formation fluids and the elevated temperature in the reservoir exceed the validity limits of commonly available thermodynamic databases needed for predictive geochemical modelling. In particular, it is shown that the activity model of Pitzer has to be applied, even if necessary input data for this model are incomplete or inconsistent for complex systems and for the considered temperatures. Simulations based on Debye- Hu¨ckel activity model lead to severe, systematic discrepancies already in the simple proposed reference case where experimental data could be used for comparison. A simplified geochemical model, consistent with the average measured composition of formation fluids and the prevailing mineralogical assemblage of the host rock, identifies the mineral phases most likely to be considered at equilibrium with the formation fluid. The simulated reactions due to CO2 injection, under the hypothesis of local thermodynamical equilibrium, result in a moderate reactivity of the system, with the dissolution of anhydrite cementation and haematite being the most relevant expected mineral reactions. This is compensated, at equilibrium, by the precipitation of new carbonates, like calcite and siderite, for an overall very small loss of porous space. The simulated rather small effect of mineral alteration is also due to the scarce amount of water available for reactions in the reservoir. The results of the model are qualitatively in line with observations from batch experiments and from natural analogues.

Phosphoenolpyruvate carboxykinase catalyzes the reversible decarboxylation of oxaloacetic acid with the concomitant transfer of the gamma-phosphate of GTP to form PEP and GDP as the first committed step of gluconeogenesis and glyceroneogenesis. The three structures of the mitochondrial isoform of PEPCK reported are complexed with Mn2+, Mn2+-PEP, or Mn2+-malonate-Mn2+ GDP and provide the first observations of the structure of the mitochondrial isoform and insight into the mechanism of catalysis mediated by this enzyme. The structures show the involvement of the hyper-reactive cysteine (C307) in the coordination of the active site Mn2+. Upon formation of the PEPCK-Mn2+-PEP or PEPCK-Mn2+-malonate-Mn2+ GDP complexes, C307 coordination is lost as the P-loop in which it resides adopts a different conformation. The structures suggest that stabilization of the cysteine-coordinated metal geometry holds the enzyme as a catalytically incompetent metal complex and may represent a previously unappreciated mechanism of regulation. A third conformation of the mobile P-loop in the PEPCK-Mn2+-malonate-Mn2+ GDP complex demonstrates the participation of a previously unrecognized, conserved serine residue (S305) in mediating phosphoryl transfer. The ordering of the mobile active site lid in the PEPCK-Mn2+-malonate-Mn2+ GDP complex yields the first observation of this structural feature and provides additional insight into the mechanism of phosphoryl transfer.

Yeast pyruvate kinase (YPK) is regulated by intermediates of the glycolytic pathway [e.g., phosphoenolpyruvate (PEP), fructose 1,6-bisphosphate (FBP), and citrate] and by the ATP charge of the cell. Recent kinetic and thermodynamic data with Mn(2+)-activated YPK show that Mn(2+) mediates the allosteric communication between the substrate, PEP, and the allosteric effector, FBP [Mesecar, A., and Nowak, T. (1997) Biochemistry 36, 6792, 6803]. These results indicate that divalent cations modulate multiligand interactions, and hence cooperativity with YPK. The nature of multiligand interactions on YPK was investigated in the presence of the physiological divalent activator Mg(2+). The binding interactions of PEP, Mg(2+), and FBP were monitored by fluorescence spectroscopy. The binding data were subject to thermodynamic linked-function analysis to determine the magnitudes of the multiligand interactions governing the allosteric activation of YPK. The two ligand coupling free energies between PEP and Mg(2+), PEP and FBP, and FBP and Mg(2+) are 0.88, -0.38, and -0.75 kcal/mol, respectively. The two-ligand coupling free energies between PEP and Mn(2+) and FBP and Mn(2+) are more negative than those with Mg(2+) as the cation. This indicates that the interactions between the divalent cation and PEP with YPK are different for Mg(2+) and Mn(2+) and that the interaction is not simply electrostatic in nature, as originally hypothesized. The magnitude of the heterotropic interaction between the metal and FBP is similar with Mg(2+) and Mn(2+). The simultaneous binding of Mg(2+), PEP, and FBP to YPK is favored by 3.21 kcal/mol compared to independent binding. This complex is destabilized by 3.30 kcal/mol relative to the analogous YPK-Mn(2+)-PEP-FDP complex. Interpretation of K(d) values when cooperative binding occurs must be done with care as these are not simple thermodynamic constants. These data demonstrate that the divalent metal, which activates phosphoryl transfer in YPK, plays a key role in modulating the various multiligand interactions that define the overall allosteric properties of the enzyme.

The enzyme phosphoenolpyruvate carboxykinase (PEPCK) catalyzes the reversible conversion of oxalacetate and GTP to phosphoenolpyruvate (PEP), GDP, and CO2. PEPCK from higher organisms is a monomer, specifically requires GTP or ITP, and uses Mn2+ as the activating cation. Currently, there is no crystal structure of GTP-utilizing PEPCKs. The conformation of the bound nucleotide was determined from transferred nuclear Overhauser effects (trnOe) experiments to determine internuclear proton distances. At 600 MHz in the presence of PEPCK, nOe effects were observed between nucleotide protons. Internuclear distances were calculated from the initial rate of the nOe buildup. These distance constraints were used in energy minimization calculations to determine the conformation of PEPCK-bound GTP. The bound nucleotide has the base oriented anti to the C2'-endo(2E) ribose ring conformation. Relaxation rate studies indicate that there is an additional relaxation effect on the C1' proton upon nucleotide binding to PEPCK. Nucleotide binding to PEPCK-Mn2+ was studied by 1H relaxation rate studies, but results were complicated by long dipole-dipole distances and the presence of competing complexes. Modification of PEPCK by iodoacetamido-TEMPO leads to an inactive enzyme that is spin-labeled at cys273. The interaction of TEMPO-PEPCK with GTP allows for the measurement of nuclear distances between GTP and the spin label. The results suggest that cys273 lies near the ribose ring of the bound nucleotide, but it is too far to be implicated in direct hydrogen bonding interactions consistent with previous results [Makinen, A. L., and Nowak, T. J. Biol. Chem. (1989) 264, 12148], suggesting that cys273 does not actively participate in catalysis. Modification of PEPCK with several cysteine specific modifying agents causes no change in the ability of the enzyme to bind nucleotide as monitored by fluorescence quenching. A correlation between the size of the modifying agent and the maximal observed quenching upon saturation of the enzyme with nucleotide is observed. This suggests a mechanism for inactivation of PEPCK by cysteine modification due to inhibition of a dynamic motion that may occur upon nucleotide binding.

The nature of bonding and energetics in trivalent rare earth π-donor ligand complexes [NdC13–πL] and [NdCp2–πL]+ have been investigated theoretically by employing Hartree–Fock (HF) and density functional theory (DFT) based methods. Acetylene, ethylene, butadiene, and benzene served as model π-ligands. Geometries and binding energies are reported. The analysis presented in this study clearly indicates the essentially electrostatic character of that binding interaction in terms of a cation–π-ligand interaction. The lanthanide to π-ligand bonding was predicted to be weak, accompanied with a slight distortion of the ligand's geometry upon coordination. The role of electron correlation does not appear to be crucial in correctly predicting the metal–ligand interaction energy and the ligand distortion.

Background: Yeast pyruvate kinase (PK) catalyzes the final step in glycolysis. The enzyme therefore represents an important control point and is allosterically activated by fructose-1,6-bisphosphate (FBP). In mammals the enzyme is found as four different isozymes with different regulatory properties: two of these isozymes are produced by alternate splicing. The allosteric regulation of PK is directly related to proliferation of certain cell types, as demonstrated by the expression of an allosterically regulated isozyme in tumor cells. A model for the allosteric transition from the inactive (T) state to the active (R) state has been proposed previously, but until now the FBP-binding site had not been identified. Results: We report here the structures of PK from yeast complexed with a substrate analog and catalytic metal ions in the presence and absence of bound FBP. The allosteric site is located 40 a from the active site and is entirely located in the enzyme regulatory (C) domain. A phosphate-binding site for the allosteric activator is created by residues encoded by a region of the gene corresponding to the alternately spliced exon of mammalian isozymes. FBP activation appears to induce several conformational changes among active-site sidechains through a mechanism that is most likely to involve significant domain motions, as previously hypothesized. Conclusions: The structure and location of the allosteric activator site agrees with the pattern of alternate genetic splicing of the PK gene in multicellular eukaryotes that distinguishes between a non-regulated isozyme and the regulated fetal isozymes. The conformational differences observed between the active sites of inactive and fully active PK enzymes is in agreement with the recently determined thermodynamic mechanism of allosteric activation through a ‘metal relay' that increases the affinity of the enzyme for its natural phosphoenolpyruvate substrate.

Chicken liver phosphoenolpyruvate carboxykinase (PEPCK) was rapidly inactivated by micromolar concentrations of ferrous sulfate in the presence of ascorbate at pH 7.4. Omitting ascorbate or replacing the Fe2+ with Mn2+ or Mg2+ gives no inactivation. Mn2+, Mg2+, or Co2+ at 100-fold molar excess over Fe2+ offered complete protection from Fe2+/ascorbate-induced inactivation. The substrates PEP and GTP, but not OAA, GDP, or CO2, offered full protection from inactivation. The addition of 5 mM EDTA stopped further inactivation of the enzyme. Thermodynamic studies indicate that the inactive enzyme no longer binds Mn2+ but still had high affinity for GTP indicating that the inactivation process was specific for the metal site. A decrease in cysteine content was observed over time following PEPCK treatment with Fe2+ and ascorbate. The apparent first-order rate constant for free sulfhydryl loss (0.085 +/- 0.005 min-1) is similar to the apparent first-order rate constant for inactivation (0.067 +/- 0.005 min-1). Amino acid composition analysis revealed that cysteic acid was generated upon Fe2+/ascorbate addition to PEPCK. Native chicken liver PEPCK has an Mr of 67 kDa. SDS-PAGE of the inactivated enzyme showed the presence of two new bands at 31.7 and 35.3 kDa indicating that PEPCK was specifically cleaved at a single site. The rate of cleavage was slower than the rate of inactivation and fully inactivated enzyme was only 50% cleaved. The Fe2+/ascorbate-catalyzed inactivation was not solely due to protein cleavage. The protein fragments generated by cleavage were separated by C4 reverse phase HPLC. The cleavage exposed a new N-terminus which was identified to be the 35.3 kDa C-terminal half of PEPCK. Sequencing of the fragments indicated that the site of cleavage was between Asp296 and Ile297. These results indicate that Asp296 is involved in metal chelation. This agrees with previous studies [Hlavaty, J. J., & Nowak, T. (1997) Biochemistry 36, 3389-3403] that suggested that Asp295 and Asp296 are involved in metal binding.

Regulation of the glycolytic pathway is considered to be primarily achieved by the carbon metabolites resulting from glucose metabolism [e.g., fructose 1,6-diphosphate (FDP), phosphoenolpyruvate (PEP), and citrate] and by the ATP charge of the cell. The divalent cations (e.g., Mg2+ and Mn2+) have not been considered as having regulatory roles in glycolysis, although they are involved in almost every enzyme-catalyzed reaction in the pathway. Using a kinetic linked-function analysis of steady-state kinetic data for the interactions of PEP, FDP, and Mn2+ with yeast pyruvate kinase (YPK), we have found that the divalent metal is the principal trigger of the allosteric responses observed with this enzyme. The interaction of Mn2+ to YPK enhances the interaction of FDP by -1.6 kcal/mol and the interaction of PEP by -2.8 kcal/mol. The simultaneous interaction of all three of these ligands to YPK is favored by -4.3 kcal/mol over the sum of their independent binding free energies. Surprisingly, the binding of the allosteric activator FDP does not directly influence the binding of the substrate PEP since a coupling free energy near zero was calculated for these two ligands. Thus, communication between the PEP and FDP sites occurs structurally through the metal by an allosteric relay mechanism. These conclusions are supported by results of a thermodynamic linked-function analysis of direct binding data for the interactions of PEP, FDP, and Mn2+ with YPK [Mesecar, A. D., & Nowak, T. (1997) Biochemistry (following paper in this series)]. Our findings raise important questions as to the possible roles of divalent metals in modulating multiligand interactions with YPK and in the regulation of the glycolytic pathway.

The influence of pH on the activation of yeast enolase by Mn2+ was measured by steady-state kinetics. The pH influence on the binding of Mn2+ to apoenolase and the enolase-substrate complex was measured by EPR spectroscopy. At pH values above 6.6, activation by Mn2+ is fit by Michaelis-Menten kinetics, but at higher concentrations of Mn2+, inhibition is observed. Under conditions analogous to the kinetic studies, the enzyme binds two Mn2+ per dimer with a Kd in the micromolar range. In the presence of the substrate 2-phosphoglycerate, three thermodynamically distinct cation binding sites per monomer are detected and the binding constants are determined by a fit to the data. As the pH decreases, the reaction velocity decreases and the cation inhibition becomes minimal. Under these conditions, only two Mn2+ binding sites per monomer are observed; the third site must be the inhibitory site. The velocity and kinetic constants are minimally affected by buffer except at pH 5.8 with PIPES. Under these conditions, the velocity is only about 40% that observed with other buffers and only a single binding site for Mn2+ per monomer is detected in the presence or absence of substrate. A direct role in the catalytic mechanism by the second cation is called to question. The binding constant for Mn2+ at site I is independent of pH over the range from 7.5 to 5.2, and the binding at site II increases only slightly over this same pH range. These results indicate that the cation sites at positions I and II contain ligands that are pH independent over this range.(ABSTRACT TRUNCATED AT 250 WORDS)

We have developed a procedure which allows isolation of virus-coded nonstructural (NS) proteins from BHK cells infected with the West Nile (WN) flavivirus in a state of purity which allows their chemical characterization. A crude membrane fraction proved to be a suitable starting material. Incubation of crude membranes in buffer containing 1.2 M guanidine hydrochloride (GH) allows isolation of weakly washed membranes. Incubation of weakly washed membranes in the presence of either 5 M GH or 8 M urea allows the isolation of stringently washed membranes, as well as a soluble protein-containing wash which is called the differential wash. Stringently washed membranes contain proteins with apparent molecular weights of 14, 19, 23, 29, and 50 kDa as predominant constituents. These proteins are of sufficient purity after SDS-PAGE to allow amino-terminal sequence determination. Together with the genome RNA sequence these analyses show that these molecules represent the virus-coded proteins NS 2b, NS 2a, pre M, NS 4b, and E, respectively. Similar analysis of the proteins present in the differential wash shows that the proteins NS 5, NS 3, and NS 1 are major constituents of this material. The carboxy-terminal sequences of NS 5 and NS 1 have also been determined.

The nature of the proton donor to the C-3 of the enolate of pyruvate, the intermediate in the reaction catalyzed by yeast pyruvate kinase, was investigated by site-directed mutagenesis and physical and kinetic analyses. Thr-298 is correctly located to function as the proton donor. T298S and T298A were constructed and purified. Both mutants are catalytically active with a decrease in k(cat) and k(cat)/K(m)(,PEP). Mn(2+)-activated T298S and T298A do not exhibit homotropic kinetic cooperativity with phosphoenolpyruvate (PEP) in the absence of fructose 1,6-bisphosphate, although PEP binding to enzyme-Mn(2+) is cooperative. The pH dependence of k(cat) for T298A indicates the loss of pK(a)(,2) = 6.4-6.9. Thr-298 affects the ionization (pK(a) approximately 6.5) responsible for modulation of k(cat). Fluorescence studies show altered dissociation constants of ligands to each enzyme complex upon Thr-298 mutations. The rates of the phosphoryl transfer and proton transfer steps in the pyruvate kinase-catalyzed reaction are altered; pyruvate enolization is affected to a greater extent. Proton inventory studies demonstrate solvent isotope effects on k(cat) and k(cat)/K(m)(,PEP). Fractionation factors are metal-dependent and significantly

Biochemical and metabolic data lead to the conclusion that the enzyme phosphoenolpyruvate carboxykinase (PEPCK) contributes to a critical point of divergence in energy conservation pathways between mammals and nematodes. The Ascaris suum PEPCK shares considerable homology with PEPCK from avian liver and is a good candidate for mutagenesis studies. The Cys306 substitution by Ser and Ala produced active enzymes and the two mutants are kinetically indistinguishable from each other. This substitution affects the catalytic affinity for the formation of the specific enzyme-nucleotide complex (k(cat)/K(m)) in the forward and reverse reactions. Studies with the substrate analogs 2(')dGDP and 2(')dGTP indicate that Cys306 in A. suum PEPCK is one of the residues important in nucleotide binding and may interact with the 2(')OH group in the ribose ring. Alternatively, mutation of this residue could cause protein changes that interfere with the proper conformation of the nucleotides for optimal catalysis to take place.

A short survey about quantum chemical descriptions of some polymerization reactions using transition metal complexes is collected in the introduction. In the case of mono-olefins the insertion reaction of the monomer takes place into the metal carbon σ-bond. For butadiene we investigated the insertion into a η3-coordinated π-allyl head group of the growing polymer chain. For the catalytic reactions the unsaturated molecule is bonded to the coordination sphere of the metal; due to the reorganization of the electronic structure the molecules becomes “activated” for the insertion step. The structure of ethylene, allyl and butadiene π-complexes with Ni° and Ni2+ was optimized with Density Function Theory (DFT) and traditional post-Hartree-Fock methods (MP2, MP4 and CCSD). The different kinds of coordinative interaction between the olefins and the metal can been discussed by the role of the frontier orbitals of a particular symmetry. Both components of the donation and back-donation bond were characterized by Charge Density Analysis (CDA) and Natural Bond Orbital (NBO) populations as well. In case of the butadiene ligand the s-cis and s-trans conformer and the transition state of their rearrangement were localized.

The pH dependence of the chemical shifts of the 31P resonances of enzyme-bound substrates 2-phosphoglycerate (PGA) and phosphoenolpyruvate (PEP) were measured to obtain further insight into the catalytic mechanism of yeast enolase. The 31P resonances of PGA and PEP bound to the enolase-Mg complex are individually observed by NMR. The Keq,internal = 1.5 favoring PEP was measured. A pH dependence of the 31P chemical shifts gives pKa values of 5.82 and 6.16 for bound PGA and PEP, respectively, indicating that both ligands bind predominantly with their phosphate groups as the dianionic species and their ionization has been altered. The phosphoryl group of PGA has been suggested as playing a role in catalysis [Nowak, T., Mildvan, A. S., and Kenyon, G. L. (1973) Biochemistry 12, 1690-1701]. The pH dependence of the kinetic parameters for Mg-enolase shows a single break in the plot of pKm, PGA vs pH at pH 6.27 with a pH independence above pH 7. This is consistent with the trianion of PGA preferably binding to the enzyme. The kcat profile gives pKA values of 5.94 and 8.35, and kcat/Km profiles give pKA values of 5.85, 6.25, and 8.39. Activation studies with Mg2+ show a pH independence for the activator constant (Ka), but a pH-dependent inhibition at higher concentrations of Mg2+. The log kcat and kcat/Ka profiles from Mg2+ activation give pKA values of about 5.9 and 8.4. These results confirm the importance of residues with pKA values of about 5.9 and 8.4 (His and Lys residues?) but do not support a function for the phosphoryl group of the substrate. The pH dependence of the Ki,Mg2+ gives pKA fits of 5. 95, 7.13, and 8.35. Data from cation inhibition suggest that the phosphate of the substrate and a His residue on enolase may bind the inhibitory Mg2+.

A new, more gentle enzyme purification for yeast enolase was developed. A series of kinetic experiments was performed with yeast enolase where the concentration of Mg(II) is kept constant and at the Km' level; the addition of Mn(II), Zn(II), or Cu(II) gives a hyperbolic decrease in the enzyme activity. The final velocity of these mixed-metal systems is the same as the velocity obtained only with Mn(II), Zn(II), or Cu(II), respectively. The concentration of the second metal that gives half-maximal effect in the presence of Mg(II) is approximately the same as the apparent Km (Km') value measured for that cation alone. Direct binding of Mn(II) to apoenolase in the absence and presence of Mg(II) shows that Mn(II) and Mg(II) compete for the same metal site on enolase. In the presence of D-2-phosphoglycerate (PGA) and Mg(II), only a single cation site per monomer is occupied by Mn(II). Water proton relaxation rate (PRR) studies of enzyme-ligand complexes containing Mn(II) and Mn(II) in the presence of Mg(II) are consistent with Mn(II) binding at site I under both conditions. PRR titrations of ligands such as the substrate PGA or the inhibitors orthophosphate or fluoride to the enolase-Mn(II)-Mg(II) complex are similar to those obtained for the enolase-Mn(II) complex, also indicating that Mn(II) is at site I in the presence of Mg(II). High-resolution 1H and 31P NMR was used to determine the paramagnetic effect of enolase-bound Mn(II) on the relaxation rates of the nuclei of the competitive inhibitor phosphoglycolate. The distances between the bound Mn(II) and the nuclei were calculated.(ABSTRACT TRUNCATED AT 250 WORDS)

The participation of lysine in the catalysis by avian liver phosphoenolpyruvate carboxykinase was studied by chemical modification and by a characterization of the modified enzyme. The rate of inactivation by 2,4-pentanedione is pseudo-first-order and linearly dependent on reagent concentration with a second-order rate constant of 0.36 +/- 0.025 M-1 min-1. Inactivation by pyridoxal 5'-phosphate of the reversible reaction catalyzed by phosphoenolpyruvate carboxykinase follows bimolecular kinetics with a second-order rate constant of 7700 +/- 860 M-1 min-1. A second-order rate constant of inactivation for the irreversible reaction catalyzed by the enzyme is 1434 +/- 110 M-1 min-1. Treatment of the enzyme with pyridoxal 5'-phosphate gives incorporation of 1 mol of pyridoxal 5'-phosphate per mole of enzyme or one lysine residue modified concomitant with 100% loss in activity. A stoichiometry of 1:1 is observed when either the reversible or the irreversible reactions catalyzed by the enzyme are monitored. A study of kobs vs pH suggests this active-site lysine has a pKa of 8.1 and a pH-independent rate constant of inactivation of 47,700 M-1 min-1. The phosphate-containing substrates IDP, ITP, and phosphoenolpyruvate offer almost complete protection against inactivation by pyridoxal 5'-phosphate. Modified, inactive enzyme exhibits little change in Mn2+ binding as shown by EPR. Proton relaxation rate measurements suggest that pyridoxal 5'-phosphate modification alters binding of the phosphate-containing substrates. 31P NMR relaxation rate measurements show altered binding of the substrates in the ternary enzyme.Mn2+.substrate complex. Circular dichroism studies show little change in secondary structure of pyridoxal 5'-phosphate modified phosphoenolpyruvate carboxykinase. These results indicate that avian liver phosphoenolpyruvate carboxykinase has one reactive lysine at the active site and it is involved in the binding and activation of the phosphate-containing substrates.

Structural analogs of the substrate oxalacetate were examined as potential substrates and inhibitors for chicken liver mitochondrial phosphoenolpyruvate (P-enolpyruvate) carboxykinase. Steady-state kinetics were employed to characterize the inhibitory effects of these substrate analogs with the enzyme. Assays were carried out in both carboxylation and decarboxylation reaction directions. Pyruvate, beta-hydroxypyruvate, beta-mercaptopyruvate, beta-fluoropyruvate, DL-lactate, glycolate, glycoaldehyde, glyoxylate, glyphosate, and DL-aspartate showed no inhibitory effects by steady-state kinetics. Oxalate, acetopyruvate, and DL-, D-, and L-glycerate exhibited weak noncompetitive inhibition of the P-enolpyruvate carboxykinase-catalyzed reaction. DL-3-Nitro-2-hydroxypropionic acid, 3-nitro-2-oxopropionic acid, DL-malate, malonate, tartronate, and alpha-ketobutyrate all show weak inhibition with estimated inhibition constants greater than 20 nM. Several of these compounds were investigated by 31P NMR to determine if they function as phosphoryl acceptors for GTP. None of the compounds tested act as phosphoryl acceptors in the enzyme-catalyzed reaction. Chicken liver mitochondrial phosphoenolpyruvate carboxykinase shows a remarkably high degree of specificity at the binding site of oxalacetate.

The modification of avian phosphoenolpyruvate carboxykinase by a variety of sulfhydryl reagents leads to inhibition. The inhibition is related to the loss of 1 highly reactive cysteine residue of the 13 cysteines present in the enzyme. Inhibition by reagents which yield a mixed disulfide was rapidly reversed by thiols. Reagents specific for vicinal sulfhydryl configurations were not potent inhibitors. The cysteine-modified enzyme continues to bind Mn2+ with the same stoichiometry and dissociation constant as the native enzyme. All of the substrates also bind to thiol-modified inactive enzyme. The modification of the reactive cysteine with the spin-labeled iodoacetate derivative leads to inactive enzyme with spin label stoichiometrically incorporated. The EPR spectrum showed an immobilized spin label on the enzyme. EPR studies of the perturbation of the phosphoenolpyruvate carboxykinase-bound spin label by bound Mn2+ showed a dipolar interaction between the two spins, estimated to be 10 A apart. The perturbation of the 1/T1 and 1/T2 values of the 31P resonances of ITP by spin-labeled enzyme indicates that this portion of the nucleotide binds 8-10 A from the spin label. These results indicate that the reactive cysteine is close to but not at the active site of the enzyme. The thiol group must be free and in its reduced form for the enzyme to be active. Perhaps modification of this group prevents conformational change(s) upon ligand binding necessary for the catalytic process.

The interactions of the substrate phosphoenolpyruvate and the substrate analogues (Z)-phosphoenol-alpha-ketobutyrate and (E)-phosphoenol-alpha-ketobutyrate with the enzyme-Mn complex of chicken liver phosphoenolpyruvate carboxykinase have been investigated by 1H and by 31P nuclear relaxation rate studies. Studies of the 1H and the 31P relaxation rates of the ligands in the binary Mn-ligand complexes show that these ligands interact with the metal ion via the phosphate group but not through the carboxylate. An inner sphere coordination complex is formed but the metal-ligand complex is not in the most extended conformation. In the relaxation rate studies of the ligands in the presence of the enzyme, conditions were adjusted so that all of the Mn2+ that was added resided in the ternary enzyme-Mn-ligand complex. The 1H relaxation rates for each of the three ligands were measured at 100 and at 300 MHz. In each case the normalized paramagnetic effects showed that 1/(pT2p) was greater than 1/(pT1p). A frequency dependence of the 1/(pT1p) and 1/(pT2p) values was also measured. The correlation time, tau c, for the Mn-1H interaction was calculated from the frequency dependence of 1/(pT1p) assuming a maximal frequency dependence of tau c and assuming no frequency dependence of tau c and from the T1M/T2M ratios at each frequency. The tau c values for all of the complexes, calculated at 100 MHz, varied from approximately 0.3 to 2.0 ns. These values were used to calculate the Mn-1H distances in each of the ternary complexes. The relaxation rates of 31P were also measured.(ABSTRACT TRUNCATED AT 250 WORDS)

The exchange inert coordination complexes, Cr(H 2 O) 4 GDP, Cr(H 2 O) 4 GTP, Cr(NH 3 ) 4 GDP, Cr(NH 3 ) 4 GTP, Co(NH 3 ) 4 GDP, and Co(NH3) 4 GTP have been synthesized and characterized. The Λ and Δ coordination isomers of Cr(H 2 O) 4 GDP, Cr(NH 3 ) 4 GDP, and the four Cr(H 2 O) 4 GTP isomers have been separated by reverse phase HPLC and characterized by their CD spectra. While the isomers of Co(NH 3 ) 4 GTP have not been successfully separated, 31 P NMR spectroscopy reveals the presence of the Λ and Δ forms. The complexes, Cr(H 2 O) 4 GDP, Co(NH 3 ) 4 GDP, Cr(H 2 O) 4 GTP, and Co(NH 3 ) 4 GTP, are linear competitive inhibitors of avian phosphoenolpyruvate carboxykinase. The K i values of 30 μM, 540 μM, 40 μM, and 12 μM, respectively, were determined for these complexes using Mn-IDP as the nucleotide substrate in the phosphoenolpyruvate carboxylation direction or Mn-ITP as nucleotide substrate for the oxalacetate decarboxylation reaction. The Λ and Δ isomers of Cr(H 2 O) 4 GDP show little specificity (a twofold maximum difference in K i ) for the enzyme. The isomeric forms of Cr(H 2 O) 4 GTP demonstrate no observed stereoselectivity of interaction with the enzyme. All of the complexes tested, except for Cr(NH 3 ) 4 GDP and Co(NH 3 ) 4 GDP, which have larger K i values, are good substrate analogs for P -enolpyruvate carboxykinase. When the substrate is Mn-GTP, fixed at 0.2 mM at pH 6.0, enzyme activity is stimulated two- to two and a half-fold by Cr(H 2 O) 4 GTP. A Dixon plot reveals that the stimulatory effect is saturated at 0.4 mM Cr(H 2 O) 4 GTP. The interaction of the enzyme with Cr(H 2 O) 4 GTP appears to produce a “memory” effect which is manifest with guanosine nucleotide substrates, but which is not observed with the alternative substrate Mn-ITP.

Phosphoenolpyruvate carboxykinase has been purified from homogenates of Ascaris suum muscle strips to apparent homogeneity as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purification is a three-step procedure which yields pure enzyme in milligram quantities with good yield. The subunit molecular weight of the Ascaris enzyme is between 75,000 and 80,000. The native molecular weight is 83,000 as determined by gel filtration. The kinetic constants for substrates of the carboxylation reaction were determined and compared to those measured for the avian liver enzyme. From kinetic studies it appears likely that two separate roles for divalent metal ions exist in the catalytic process. Studies conducted with Mn2+ or with micromolar concentrations of Mn2+, in the presence of millimolar concentrations of Mg2+ suggest that Mn2+ but not Mg2+ binds directly to and activates the enzyme while either Mn2+ or Mg2+ may bind to the nucleotide resulting in the metal-nucleotide complex. The metal-nucleotide is the active form of the substrate for the reaction. In the presence of Mg2+, an increase in the Mn2+ concentration results in a decrease in the Km for P-enolpyruvate suggesting a direct role for Mn2+ stimulation and regulation of activity. The concentrations of Mn2+ and Mg2+ in Ascaris muscle strips were determined by atomic absorption spectroscopy and support the proposed hypothesis of a specific Mn2+ activation of the enzyme. The nucleotides ATP and ITP act as competitive inhibitors against GTP with KI values of 0.50 and 0.75 mM, respectively. ITP is a competitive inhibitor against both IDP and P-enolpyruvate, suggesting overlapping binding sites for the two substrates on the enzyme.

The interactions of nucleotides with phosphoenolpyruvate carboxykinase were studied by using the stereospecific thiophosphate analogues of GDP and GTP. The metal ion dependent stereoselectivity of these analogues was determined by using steady-state kinetics. The RP and SP isomers of guanosine 5'-O-(1-thiodiphosphate) (GDP alpha S) were substrates with low turnover, and a small preference for the RP isomer was observed. Neither the enzyme-metal nor the nucleotide-metal complex elicited any substantial change in the selectivity. Guanosine 5'-O-(2-thiodiphosphate) (GDP beta S) exhibited no substrate activity for the enzyme, regardless of the cations. This nucleotide was a competitive inhibitor against GDP, however. Both RP and SP diastereomers of guanosine 5'-O-(1-thiotriphosphate) (GTP alpha S) were good substrates for phosphoenolpyruvate carboxykinase; in several cases, depending upon the cation, kcat and/or Vm/Km for the RP isomer is greater than for the substrate GTP. The enzyme-metal complex but not the nucleotide-metal complex affects the relative Km and the Vmax values. In contrast, guanosine 5'-O-(2-thiotriphosphate) (GTP beta S) (SP) is a much better substrate (greater than 50 times) than is GTP beta S (RP). The metal ions have little effect on the selectivity. These results suggest a specific interaction of the beta-phosphate of the nucleotide with the protein. The analogue guanosine 5'-O-(3-thiotriphosphate) (GPT gamma S) serves as a substrate to yield GDP and thiophosphoenolpyruvate. The latter was detected by 31P NMR and was shown to slowly hydrolyze to form phosphoenolpyruvate.(ABSTRACT TRUNCATED AT 250 WORDS)

Formyltetrahydrofolate synthetase, a tetrameric protein, contains 23 half-cystine residues as determined by cysteic acid analysis and 24 free sulfhydryl groups as determined by four different methods thus indicating absence of disulfide bonds. All of the cysteine residues react with sulfhydryl reagents in the absence of denaturing agents. Titration of the enzyme with p-chloromercuribenzoate (CMB) caused a loss in activity which was a linear function of the amount of thiol groups reacted. A complete loss in activity occurred after 11 CMB molecules had reacted per mole of enzyme. The loss in activity was accompanied by the appearance of the monomeric form of the protein; however, the presence of tetramer which contained CMB was demonstrated by Sephadex G-200 chromatography of enzyme which had been partially inactivated with 14C-labeled CMB. The reaction of the enzyme with 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) was different from that with CMB. The relation between thiol groups reacted and loss in activity was not linear, and about 18 to 20 thiol groups had to react before a complete loss in activity occurred. Dissociation to the monomeric form did not occur until about 75% of the cysteine residues had reacted. Titration with HgCl2 showed that a linear decrease in activity occurred until complete inactivation was observed after 8 Hg2+ ions had been bound by the protein. Hg2+ could be bound to virtually all of the cysteine residues without causing dissociation of the enzyme. Reactivation by cysteine of enzyme inactivated by CMB or DTNB was variable and could be achieved only when the enzyme was still in the tetrameric form. Total reactivation of mercury enzyme was achieved with 2-mercaptoethanol or cysteine. The inactivation by CMB and DTNB was retarded by ATP and tetrahydrofolate and facilitated by MgATP. Formate and Mg2+ had no effect on the inactivation. It is concluded that the inactivation of formyltetrahydrofolate synthetase by sulfhydryl reagents results from conformational changes produced in the tetramer as well as from dissociation of the tetramer to the inactive monomer. The effects produced by the substrates probably result from conformational changes in the protein. There is no evidence which suggests that a sulfhydryl group or groups constitute part of the active site of the enzyme.

Pyruvate kinase, which has an absolute requirement for both a monovalent and a divalent cation for activity, can be activated by the monomethylammonium cation at a rate 0.5% that measured in the presence of K+. The kinetically determined dissociation constant for the monovalent activator in the presence of saturating substrates (K1 = 85 mm) agrees with that measured for K+ and the dissociation constant measured in the presence of saturating substrates and the divalent activator Mn2+, (K4 = 20.5 ± 3.3 mm) is somewhat larger than that for K+. The presence of carbonbound protons on the movovalent activator permits the investigation of the interaction of the monovalent activator with the enzyme-Mn2+ complexes by proton nuclear magnetic resonance techniques. The presence of the enzyme has no effect on the longitudinal (1/T1) and transverse (1/T2) relaxation rates of the carbon-bound protons. The formation of the enzyme-Mn2+ complex increases the relaxation rates due to the electron-nuclear dipolar interaction of the paramagnetic Mn2+ and the protons. In the presence of the substrate phosphoenolpyruvate, the relaxation rates are even further increased. A measure of the relaxation rates at two frequencies allows the evaluation of the correlation time for the electron-nuclear interaction, τc by several methods. No significant change in the correlation time is seen when the substrate complex is formed. The electron-nuclear distance can thus be evaluated from the correlation time, τc, and from the values measured for 1/T1. In the absence of substrate the protons of the methyl group are 8.3 ± 0.6 A from the enzyme-bound Mn2+. Upon formation of the pyruvate kinase-Mn2+-phosphoenolpyruvate complex, the protons move to 6.6 ± 0.2 A of the Mn2+. This result shows that the substrate affects the conformation of the active site by bringing the monovalent and divalent cation activators in closer proximity and that the monovalent activator can indeed bind in the active site of pyruvate kinase. The Mn2+-methyl proton distance measured is that predicted if the monovalent cation acts by binding P-enolpyruvate via the carboxyl group to form the active complex (Nowak, T., and Mildvan, A. S. (1972) Biochemistry 11, 2819). Monomethylammonium ion can thus serve as a useful probe in studying the interactions of monovalent cations with many biological systems with relative ease and should lead to a greater understanding of monovalent cation activation and function in biology.

31P-NMR has been applied to the study of the metabolisms of the intact parasitic helminths Ascaris suum (the intestinal roundworm) and Fasciola hepatica (the liver fluke). After calibration of the chemical shift of Pi in muscle extracts the internal pH of adult Ascaris worms and the effect of the pH of the external medium on the organism's internal pH were measured. Assignments of nearly all of the observable 31P resonances could be made. A large resonance from glycerophosphorylcholine whose function is unclear was observed but no signals from energy storage compounds such as creatine phosphate were detected. The profiles of the phosphorus-containing metabolites in both organisms were monitored as a function of time. Changes in sugar phosphate distributions but not ATP/ADP were observed. Studies of the drug closantel on Fasciola hepatica were performed. Initial effects of the drug were a decrease in glucose 6-phosphate and an increase in Pi with no substantial change in ATP levels as observed by 31P-NMR. Studies involving treatment with closantel followed by rapid freezing, extraction, and analytical determination of glycolytic intermediates confirmed NMR observations. This NMR method can serve as a simple noninvasive procedure to study parasite metabolism and drug effects on metabolism.

The proteolytic processes involved in the synthesis of the structural proteins of the West Nile (WN) flavivirus were analyzed: The carboxy-terminal sequences of the structural proteins were determined and the proteins translated in vitro in the presence of membranes from a mRNA coding for the structural polyprotein were analyzed. The results obtained indicate that the following proteolytic activities are involved in the synthesis and assembly of WN virus structural proteins: The growing peptide chain which contains the sequences of the structural proteins in the order C-pre-M-E is cleaved at three places by cellular signalase(s). This cleavage generates the primary amino acid sequence of the mature structural proteins pre-M and E (and the amino-terminus of the ensuing nonstructural protein NS 1). The amino-terminal part of the polyprotein containing the amino acid residues 1 to 123 is released as a molecule which migrates slightly slower than the mature viral core protein and which presumably is associated to the RER membranes via its carboxy-terminal sequence. This protein is called the anchored C protein. Priorto or during assembly of intracellular virus particles the anchored C protein is converted into mature C protein by removal of the carboxy-terminal hydrophobic segment containing the amino acid residues 106 to 123. Presumably a virus-coded protease which can cleave the polyprotein after two basic amino acid residues is responsible for this cleavage. The cell-associated WN virus particles are constructed from the proteins C, pre-M, and E which contain the amino residues 1–105, 124–290, and 291–787 of the polyprotein, respectively. Cleavage of the pre-M protein between amino acid residues 215 and 216, presumably by a cellular enzyme located in the Golgi vesicles, and loss of the amino-terminal fragment of this protein are associated with the release of virus from the cells.