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PubMed1997

Biogenesis of L-glyceric aciduria, oxalosis and renal injury in rats simulating type II primary hyperoxaluria.

Tracer experiments in rats mimicking type II primary hyperoxaluria, with an expanded intracellular pool of hydroxypyruvate, showed that the excess formation of oxalate did not originate from its immediate precursor glyoxylate. In these animals, the hepatic and kidney activities of oxalate synthesising enzymes such as lactate dehydrogenase and glycolate oxidase were normal, but tissue lipid peroxidation was significantly higher. In vitro experiments established that in a mild alkaline solution, hydroxypyruvate underwent auto-oxidation to form oxalate and H2O2 and also inhibited lactate dehydrogenase and glycolate oxidase from oxidising glyoxylate to oxalate. On the basis of the experimental evidence, we suggest that in type II primary hyperoxaluria, the accumulating hydroxypyruvate could reduce the intracellular pool of glyoxylate and on ageing, give rise to excess oxalate and H2O2, to cause oxalosis in the former and free radical mediated-cell injuries in the latter.

Animals↗

PubMed1993

A role for the epsilon-amino group of lysine-334 of ribulose-1,5-bisphosphate carboxylase in the addition of carbon dioxide to the 2,3-enediol(ate) of ribulose 1,5-bisphosphate.

Earlier structural and functional studies of ribulose-1,5-bisphosphate carboxylase/oxygenase imply that K334 facilitates the addition of gaseous substrate to the 2,3-enediol(ate) derived from ribulose 1,5-bisphosphate. Crystallographic analysis of the activated spinach enzyme [Knight et al. (1990) J. Mol. Biol. 215, 113-160] shows that the lysyl side chain is appropriately positioned to stabilize the transition state for the addition of CO2 to the enediol(ate). Furthermore, despite total impairment of carboxylase and oxygenase activities, site-directed mutants of the Rhodospirillum rubrum enzyme with replacements for lysine K334 (formerly designated K329) retain the capacity to enolize ribulose bisphosphate, demonstrating that the primary catalytic lesion lies beyond this initial step [Soper et al. (1988) Protein Eng. 2, 39-44; Hartman & Lee (1989) J. Biol. Chem. 264, 11784-11789]. We now show that the K334C mutant is also competent in the latter stages of catalysis, whereby 2'-carboxy-3-keto-D-arabinitol 1,5-bisphosphate, the six-carbon intermediate of the carboxylation pathway, is correctly processed to 3-phosphoglycerate. Thus, the impairment of the mutant in overall catalysis can be attributed to preferential disruption of the reaction of CO2 or O2 with the enzyme-bound enediol(ate). Chemical rescue of the K334C mutant by aminoethylation and aminopropylation shows that this disruption reflects, at least in part, a failure to adequately stabilize the relevant transition state. With several simplifying assumptions, the CO2/O2 specificity factor tau can be reduced to the ratio of the fundamental second-order rate constants for the interaction of the gaseous substrates with the enzyme-bound 2,3-enediol(ate) of ribulose bisphosphate.(ABSTRACT TRUNCATED AT 250 WORDS)

Binding Sites↗

PubMed1992

1H and 51V NMR studies of the interaction of vanadate and 2-vanadio-3-phosphoglycerate with phosphoglycerate mutase.

The formation of complexes of vanadate with 2-phosphoglycerate and 3-phosphoglycerate have been studied using 51V nuclear magnetic resonance spectroscopy. Signals attributed to two 2,3-diphosphoglycerate analogues, 2-vanadio-3-phosphoglycerate and 2-phospho-3-vanadioglycerate, were detected but were not fully resolved from signals of inorganic vanadate and the anhydride formed between vanadate and the phosphate ester moieties of the individual phosphoglycerates. Equilibrium constants for formation of the two 2,3-bisphosphate analogues were estimated as 2.5 M-1 for 2-vanadio-3-phosphoglycerate and 0.2 M-1 for 2-phospho-3-vanadioglycerate. The results of the binding study are fully consistent with non-cooperativity in the binding of vanadiophosphoglycerate to the two active sites of phosphoglycerate mutase (PGM). 2-Vanadio-3-phosphoglycerate was found to bind to the dephospho form of phosphoglycerate mutase with a dissociation constant of about 1 x 10(-11) M at pH 7 and 7 x 10(-11) M at pH 8. Three signals attributed to histidine residues were observed in the 1H NMR spectrum of phosphoglycerate mutase. Two of these signals and also an additional signal, tentatively attributed to a tryptophan, underwent a chemical shift change when the vanadiophosphoglycerate complex was bound to the enzyme. The results obtained here are in accord with these vanadate-phosphoglycerate complexes being much more potent inhibitors of phosphoglycerate mutase than either monomeric or dimeric vanadate. The dissociation constant of 10(-11) M for 2-vanadio-3-phosphoglycerate is about 4 orders of magnitude smaller than the Km for PGM, a result in accordance with the vanadiophosphoglycerates being transition state analogues for the phosphorylation of PGM by 2,3-diphosphoglycerate.(ABSTRACT TRUNCATED AT 250 WORDS)

Binding Sites↗

PubMed1994

Primary and secondary kinetic isotope effects as probes of the mechanism of yeast enolase.

Enolase catalyzes the interconversion of 2-phosphoglycerate and phosphoenolpyruvate. Kinetic isotope effects have been used to determine whether abstraction of the proton from C-2 and loss of hydroxide from C-3 of 2-phosphoglycerate occur in a concerted reaction or as sequential processes and whether these steps are kinetically significant for the enolase-catalyzed reaction. Enolase exhibits a significant primary deuterium isotope effect, as well as catalyzing the relatively rapid exchange of the C-2 proton with solvent water. Secondary C-3 deuterium isotope effects are also reported, both when the C-2 carbon carries a hydrogen and when this center is deuterated. These results provide information about the kinetic significance and timing of the transition state(s) associated with the loss of H+ and OH-. Strong evidence has been presented for a stepwise mechanism where both the rate of proton abstraction and one or both of the later transition states, i.e., those associated with hydroxide loss and product release, limit the overall reaction rate. If a concerted reaction were to be invoked, the presence of a small secondary 2H isotope effect in combination with the observed rate of exchange of the C-2 proton require the intrinsic secondary 2H kinetic isotope effect to be effectively unity. For the concerted mechanism, an intrinsic effect of unity would be consistent only with an extremely asymmetric transition state that is dominated by C-H bond cleavage.

Deuterium↗

PubMed1982

Binding of fluoride by yeast enolase.

The kinetics of fluoride binding by yeast enolase have been examined by direct measurement of equilibrium fluoride (F) concentrations with an ion-specific electrode. Mg2+ and inorganic phosphate (Pi) affect the binding of F, and evidence is presented for an ordered binding mechanism in which phosphate and F interact with conformational and catalytic Mg2+ species, with the overall formation of a quaternary complex. A maximum of four atoms of F were bound per enzyme dimer, and the data point to the nonequivalent binding of pairs of F atoms. The dissociation constants were 5.0 X 10(-4) M and 8.2 X 10(-5) M for the first and second pairs of F atoms, respectively. The first pair of binding sites was filled when the ratio of phosphate ions/enolase dimer exceeded 2 and appeared to involve the pair of conformation Mg2+ ions. The binding of the second pair of F atoms followed the binding of catalytic Mg2+ in the presence of Pi and, furthermore, appeared to exhibit positive cooperativity with respect to F. The data suggest, also, that the binding of Pi may involve sequential addition of Pi pairs to the different Mg2+ species on the enzyme. F binding was at a maximum between pH 5.5 and pH 6.0, consistent with an involvement of the monovalent form of Pi. In the absence of added Pi, (MgF)+ appeared to be the preferred ligand. Addition of the enzyme substrate 2-phosphoglycerate led to the release of bound F. These findings are consistent with the known patterns of inhibition of enzymatic activity by F.

Dose-Response Relationship, Drug↗

PubMed1982

Ribulose-1,5-bisphosphate carboxylase: enzyme-catalyzed appearance of solvent tritium at carbon 3 of ribulose 1,5-bisphosphate reisolated after partial reaction.

When ribulose 1,5-bisphosphate is allowed to react with carbon dioxide in tritiated water in the carboxylation reaction catalyzed by ribulose-1,5-bisphosphate carboxylase from Rhodospirillum rubrum, the ribulose 1,5-bisphosphate reisolated after partial reaction is found to be labeled. The specific radioactivity of the remaining substrate pool rises during the course of the reaction. Experiments in deuterium oxide show that the isotopic label resides on carbon 3. Earlier failures to detect this exchange process probably derive from the use of enzyme that was, in the absence of carbon dioxide, inactive. The present results provide direct evidence for the intermediacy of the enediol between C-2 and C-3 of ribulose 1,5-bisphosphate and show that the enolization step is at least partially rate limiting in the overall carboxylase reaction. The specific radioactivity of the product 3-phospho-D-glycerate remains constant throughout the course of the reaction at about one-sixth that of the solvent. This strengthens the argument against the involvement of "sticky" protons in the reaction.

Carbon↗

PubMed1985

Coordination scheme and stereochemical configuration of manganese(II) adenosine 5'-diphosphate at the active site of 3-phosphoglycerate kinase.

The structure of the MnIIADP complex at the active site of 3-phosphoglycerate kinase from yeast has been investigated by electron paramagnetic resonance (EPR) spectroscopy. Inhomogeneous broadening in the EPR signals for Mn(II) resulting from unresolved superhyperfine coupling to 17O regiospecifically incorporated into ADP shows that Mn(II) is coordinated to the alpha- and beta-phosphate groups of ADP at the active site of the enzyme. The EPR pattern for the enzyme-MnIIADP complex is characteristic of a predominantly axially symmetric zero-field splitting tensor. The symmetry and magnitude of the zero-field splitting interaction suggest that there is an additional negatively charged oxygen ligand in the coordination sphere of Mn(II). EPR measurements for solutions of the enzyme-MnIIADP complex in 17O-enriched water indicate that there are also two or three water molecules in the coordination sphere of the metal ion. EPR data for complexes with the two epimers of [alpha-17O]ADP have been used to determine the stereochemical configuration of the MnIIADP complex at the active site. EPR spectra for Mn(II) in the enzymic complex with (Rp)-[alpha-17O]ADP show an inhomogeneous broadening due to superhyperfine coupling with 17O whereas spectra for (Sp)-[alpha-17O]ADP complexes are indistinguishable from those for matched samples with unlabeled ADP. These results show that 3-phosphoglycerate kinase selectivity binds the alpha configuration of the alpha, beta chelate of MnIIADP. Addition of 3-phosphoglycerate to form the dead-end complex (enzyme-MnIIADP-3-phosphoglycerate) does not alter the EPR spectrum, but addition of vanadate to this complex causes marked changes in the spectral parameters.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Diphosphate↗

PubMed1988

31P NMR studies of enzyme-bound substrate complexes of yeast 3-phosphoglycerate kinase. 2. Structure measurements using paramagnetic relaxation effects of Mn(II) and Co(II).

Measurements of the paramagnetic effects of two dissimilar activating paramagnetic cations, Mn(II) and Co(II), on the spin relaxation rates of the 31P nuclei in the complexes of 3-phosphoglycerate kinase with ATP, ADP, and 3-P-glycerate have been used to study the structures of these enzyme-substrate complexes. All experiments were performed on enzyme-bound complexes, so that two exchanging complexes (with and without cation) contribute to the observed relaxation rate. Measurements were made at three 31P NMR frequencies, 81, 121.5, and 190.2 MHz, and as a function of temperature in the range 5-20 degrees C to determine the effect of exchange on the observed relaxation rates. Relaxation rates in E.MnADP and E.MnATP were shown to be exchange-limited, and therefore bereft of structural information, both by lack of frequency dependence and by temperature dependence with activation energies (delta E) in the range 5-8 kcal/mol. Relaxation rates for E.CoADP and E.CoATP exhibit frequency dependence and delta E values in the range 1-3 kcal/mol; i.e., these rates depend on the Co(II)-31P distances. Difficulties involved in estimating electron relaxation times in E.CoADP and E.CoATP restrict calculation of Co(II)-31P distances in these complexes to upper and lower limits. These distances were all in the range 2.7-4.1 A, appropriate for direct coordination of Co(II) to the phosphate groups.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Diphosphate↗

PubMed1990

Stereochemical course of the reactions catalyzed by the bacterial phosphoenolpyruvate:mannitol phosphotransferase system.

We have determined the overall stereochemical course of the reactions leading to the phosphorylation of D-mannitol by mannitol-specific enzyme II (EIIMtl) of the Escherichia coli phosphoenolpyruvate- (PEP) dependent phosphotransferase system (PTS). In the presence of enzyme I and HPr of the PTS, and of membranes containing EIIMtl, the phospho group from [(R)-16O,17O,18O]PEP was transferred to D-mannitol to form mannitol 1-phosphate with overall inversion of the configuration at phosphorus with respect to that of PEP. Since in the course of these reactions enzyme I and HPr are each covalently phosphorylated at a single site and inversion of the chiral phospho group from PEP indicates an odd number of transfer steps overall, transfer from phospho-HPr to mannitol via EIIMtl must also occur in an odd number of steps. Taken together with the fact that catalytically important phospho-EIIMtl intermediates have been demonstrated biochemically, our results imply that EIIMtl is sequentially phosphorylated at two different sites during phospho transfer from phospho-HPr to mannitol. This conclusion is consistent with the available evidence on phospho-EIIMtl intermediates and in particular with the recent report that two different phospho peptides can be isolated from the fully phosphorylated protein [Pas, H. H., & Robillard, G. T. (1988) Biochemistry 27, 5835-5839].

Binding Sites↗

PubMed1976

Specific, reversible inactivation of phosphofructokinase by fructose-1,6-bisphosphatase. Involvement of adenosine 5'-triphosphate, oleate, and 3-phosphoglycerate.

Optimal conditions necessary for the reversible inactivation of crystalline rabbit muscle phosphofructokinase by homogeneous rabbit liver fructose-1,6-bisphosphatase have been studied. At higher enzyme levels (to 530 mug/ml of phosphofructokinase) the two proteins were mixed and incubated in a pH 7.5 buffer composed of 50 mM Tris-HC1, 2 mM potassium phosphate, and 0.2 mM dithiothreitol. Aliquots were removed at various times and assayed for enzyme activity. A time dependent inactivation of phosphofructokinase caused by 1-2.3 times its weight of fructose-1,6-bisphosphatase was observed at 30, 23, and 0 degree C. This inactivation did not require the presence of adenosine 5'-triphosphate or Mg2+ in the incubation mixture, but an adenosine 5'-triphosphate concentration of 2.7 mM or greater was required in the assay to keep phosphofructokinase in an inactive form. A mixture of activators (inorganic phosphate, (NH4)2SO4, and adenosine 5'-monophosphate), when added to the assay cuvette, restored nearly all of the expected enzyme activity. Incubations with other proteins, including aldolase, at concentrations equal to or greater than the effective quantity of fructose-1,6-bisphosphatase had no inhibitory effect on phosphofructokinase activity. Removal of tightly bound fructose 1,6-bisphosphate from phosphofructokinase could not explain this inactivation, since several analyses of crystalline phosphofructokinase averaged less than 0.1 mol of fructose 1,6-bisphosphate/320 000 g of enzyme. Furthermore, the inactivation occurred in the absence of Mg2+ where the complete lack of fructose-1-6-bisphosphatase activity was confirmed directly. At lower phosphofructokinase concentrations (0.2-2 mug/ml) the inactivation was studied directly in the assay cuvette. Higher ratios of fructose-1,6-bisphosphatase to phosphofructokinase were necessary in these cases, but oleate and 3-phosphoglycerate acted synergistically with lower amounts of fructose-1,6-bisphosphatase to cause inactivation. The inactivation did not occur when high concentrations of fructose 6-phosphate were present in the assay, or when the level of adenosine 5'-triphosphate was decreased. However, the inactivation was found at pH 8, where the effects of allosteric regulators on phosphofructokinase are greatly reduced. Experiments with rat liver phosphofructokinase showed that this enzyme was also subject to inhibition by rabbit liver fructose 1,6-bisphosphatase under conditions similar to those used in the muscle enzyme studies. Attempts to demonstrate direct interaction between phosphofructokinase and fructose-1,6-bisphosphate by physical methods were unsuccessful. Nevertheless, our results suggest that, under conditions which approximate the physiological state, the presence of fructose-1,6bisphosphatase can cause phosphofructokinase to assume an inactive conformation. This interaction may have a significant role in vivo in controlling the interrelationship between glycolysis and gluconeogenesis.

Adenosine Triphosphate↗

PubMed1974