Phosphoglycerate kinase--glyceraldehyde-3-phosphate dehydrogenase interaction: reaction rate studies.

Rate studies using phosphoglycerate kinase (PGK)--glyceraldehyde-3-phosphate dehydrogenase (GPDH) enzyme pair have been carried out to distinguish between the two mechanisms of intermediate metabolite transfer, namely diffusion through the solvent versus "substrate channelling" within an enzyme-enzyme complex. A procedure has been described for the assay of the rates of PGK-catalysed and the PGK-GPDH coupled reactions at high (saturating) GPDH concentration. With PGKs of rabbit muscle and yeast, the coupled reaction proceeded faster than the PGK-catalysed reaction. At a high salt concentration (0.5 M KCl), where a PGK-GPDH complex is known to dissociate, the two reactions proceeded at almost equal rates. At fixed PGK concentration, the rate of the coupled reaction at high (saturating) GPDH concentration varied with the nature (biological origin) of the latter enzyme. In the presence of 0.5 M KCl, the saturating rate values with different GPDHs were almost equal. The PGK-catalysed reaction exhibited typical Michaelian behaviour on varying the substrate concentrations (linear double reciprocal plots). The Km values for 3-PGA (0.51 mM) and ATP (0.40 mM) were independent of the concentration of the second substrate. The double reciprocal plots for the coupled reaction showed downward curvature, i.e. activation at higher substrate concentrations. The ratio of the rate of the coupled reaction: the rate of the PGK catalysed reaction was found to be a function of the nature of PGK, nature of GPDH, nature of buffer, pH, salt concentration and substrate concentrations. The ratio varied between close to unity at low substrate concentrations, to three when the Vmax values of the two reactions were compared. At low substrate concentrations, the rate of the coupled reaction became independent of the nature of GPDH. It has been suggested that in the PGK-GPDH pair, the intermediate metabolite (BPG) is transferred directly from one enzyme to the other within an enzyme-enzyme complex, except at high salt or low substrate concentrations. Under the latter conditions, data were consistent with metabolite transfer by diffusion. Implications of these results for coupled enzyme assays have been discussed.

Regulatory properties and active site groups of cytosolic mung bean pyruvate kinase.

Properties of mung bean pyruvate kinase were studied and the active site groups were derived. Metabolites like AMP, glucose, glucose-6-phosphate, fructose-6-phosphate, fructose-1, 6-bisphosphate, 3-phospho-glycerate, isocitrate, malate and alpha-ketoglutarate had practically no effect on pyruvate kinase activity. Alanine, serine, glutamine, methionine and GMP had a weak activating effect on the enzyme. Some metabolites such as ATP, GTP, and UMP were found to be weakly inhibitory. Moderate to strong inhibition was observed with citrate, succinate, glutamate and oxalate. Inhibition brought about by ATP and citrate when present together showed synergistic effect. Inhibition by citrate was non-competitive with respect to both PEP and ADP suggesting the presence of a regulatory site. Mung bean pyruvate kinase showed half optimal activity at pH 6.6 and 8.9 at saturating concentrations of PEP, ADP and Mg2+. Small concentrations of the SH specific reagents, namely iodoacetamide (0.1 and 0.2 mM), N-ethylmaleimide(0.05-0.1 mM) and p-chloromercuribenzoate (0.1 mM) inactivated the enzyme; single exponential loss of activity was observed in each case. Photooxidation of the enzyme in the presence of methylene blue (100 and 200 micrograms/ml) and rose bengal (5 and 10 micrograms/ml) also led to a single exponential activity decay. When the enzyme was treated with diethyl pyrocarbonate (DEP), a time dependent exponential decay in its activity was observed with a parallel increase in absorbance at 240 nm. PEP protected the enzyme against inactivation by DEP. Reagents specific for tyrosine (iodine and tetranitromethane) and tryptophan residues (N-bromosuccinimide) residues had no effect. These observations confirm that SH and imidazole groups are vital for the activity of the enzyme.

Purification, characterisation and steady state kinetic properties of cytosolic pyruvate kinase free of phosphoenol pyruvate phosphatase activity from germinating mung beans (Vigna radiata L.)

Mung bean pyruvate kinase (PK) practically free from PEP-phosphatase has been purified about 36 fold. The enzyme is irreversibly inactivated on desalting by gel filtration or dialysis (without EDTA). The inactivation is also observed in the presence of ATP, Mg2+ or thiols but is prevented by a non-proteinous, heat stable, small molecular mass factor present in the mung bean extract. Mung bean PK has a molecular mass of 210 kDa. It shows single exponential decay of activity at various temperatures (-4 to 60 degrees C). The Km of PEP and ADP are found to be 0.12 and 0.24 mM, respectively at pH 6.5, when the enzyme is saturated with the second substrate. The Km values for PEP and ADP are 0.05 and 0.16 mM, at pH 8.5 and 0.09 and 0.17 mM, respectively at pH 7.5. The optimum pH is 7.5. The enzyme shows an absolute requirement for Mg2+ (Km 0.43 mM) or Mn2+ ions (Km 0.125 mM). Potassium ions are not essential but activate the enzyme in the presence of Mg2+ or Mn2+ ions. ATP shows competitive inhibition with ADP and non-competitive with PEP. Kinetic studies at different pHs and effects of ATP suggest the formation of a ternary complex (E.ADP.PEP) by a combination of random and compulsory ordered pathways depending on the experimental conditions.

Inactivation of glyceraldehyde-3-phosphate dehydrogenase with SH-reagents and its relationship to the protein quaternary structure.

Inactivation of mung bean glyceraldehyde-3-phosphate dehydrogenase (GPDH) with excess iodoacetate or N-ethylmaleimide exhibits pseudo-first order kinetics at pH 7.3 and 8.6 in the absence and presence of NAD+, suggesting that all the reactive SH groups (four per tetrameric GPDH molecule) have equivalent reactivity towards these reagents. This is similar to the D2-symmetry conformation proposed on the basis of thermal inactivation data [Malhotra and Srinivasan, Arch. Biochem. Biophys. 236, 775-781 (1985)]. With p-chloromercury benzoate (p-CMB), the inactivation of GPDH is very fast and its kinetics can be monitored at low reagent concentration only. Keeping a high molar p-CMB: enzyme ratio (= 47), the kinetics were found to be biphasic, with half of the activity being lost in a fast and the remaining in a slow phase, characteristic of C2-symmetry conformation and half site reactivity. The p-CMB inactivation could be largely reversed on the addition of excess cysteine. A comparison of these data with literature reports on this and other GPDHs reveals that all reagents having large non-polar moieties exhibit half site reactivity with this enzyme.

Effect of substrate and phosphate ions on the quaternary structure symmetry of glyceraldehyde-3-phosphate dehydrogenases of mung beans and rabbit muscle.

Effects of glyceraldehyde-3-phosphate (G-3-P) and phosphate ions on thermal inactivation of glyceraldehyde-3-phosphate dehydrogenases (GPDHs) of mung beans and rabbit muscle have been studied at different pH. In the absence of any ligand, the two enzymes show a striking similarity in the pH-dependence of the kinetics of thermal inactivation. At lower pH values both the enzymes biphasic kinetics with each phase accounting for about half of the starting activity (a C2 symmetry of the homotetrameric enzyme molecule). The kinetics change to a single exponential decay at higher pH values, a D2 symmetry [Malhotra & Srinivasan (1985) Arch. Biochem. Biphys. 236, 775-781; Malhotra & Tikoo (1991) Indian, J. Biochem. Biophys. 28, 16-21]. With each enzyme, phosphate ions are found to have no effect on the kinetic pattern at lower pH, but G-3-P brings about a change from biphasic to a single exponential decay. At higher pH values, G-3-P has no effect on the single exponential decay kinetic pattern, but phosphate ions change the same to a biphasic loss of activity with each phase accounting for about half of the starting activity. It has been concluded that with both the enzymes, G-3-P and phosphate ions have higher affinity and stabilise the D2- and C2-symmetry conformation, respectively. Binding isotherms of the two substrates for these enzymes have been described based on the ligand concentration-dependence of the changes in the rate constants and kinetic pattern of thermal inactivation.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of coenzymes on the quaternary structure conformation of glyceraldehyde-3-phosphate dehydrogenases of mung beans and rabbit muscle.

Kinetics of thermal inactivation of glyceraldehyde-3-phosphate dehydrogenases of mung beans and rabbit muscle have been studied under different pH conditions in the absence and presence of various concentrations of NAD+ and NADH. The data have been discussed with respect to the effect of the coenzymes on the quaternary structure symmetry of the two enzymes and their binding isotherms. Both the (homo-tetrameric) apo-enzymes exhibit biphasic kinetics of thermal inactivation, characteristic of C2 symmetry, at lower pH values and a single exponential decay of enzyme activity, characteristic of D2 symmetry, at higher pHs. In each case, NAD+ has no effect on the biphasic kinetic pattern of thermal inactivation at lower pH values, but NADH brings about a change to single exponential decay. At higher pH values, NADH does not affect the kinetic pattern (single exponential decay) of any enzyme, but NAD+ alters it to biphasic kinetics in each case. The data suggest that NAD+ and NADH have higher affinity for the C2 and D2 symmetry conformation, respectively. With mung beans enzyme, the effect of NAD+ on the two rate constants of biphasic inactivation at pH 7.3 is consistent with a Kdiss equal to 110 microM. The NAD(+)-dependent changes in the kinetic pattern of thermal inactivation of this enzyme at pH 8.6 suggest a positive cooperativity in the coenzyme binding (nH = 3.0). In the binding of NADH to the mung beans enzyme, a weak positive cooperativity is observed at pH 7.3.(ABSTRACT TRUNCATED AT 250 WORDS)

Circular dichroism studies of the coenzyme environment in the active sites of mutant forms of the beta-subunit in the tryptophan synthase alpha 2 beta 2 complex.

The circular dichroism has been used to evaluate the effect of mutation on the environment of the pyridoxal phosphate coenzyme in the active site of the beta-subunit in the tryptophan synthase alpha 2 beta 2 complex from Salmonella typhimurium. Seven mutant forms of the alpha 2 beta 2-complex with single amino acid replacements at residues 87, 109, 188, 306, and 350 of the beta-subunit have been prepared by site-directed mutagenesis, purified to homogeneity, and characterized by absorption and circular dichroism spectroscopy. Since the wild type and mutant alpha 2 beta 2 complexes all exhibit positive circular dichroism in the coenzyme absorption band, pyridoxal phosphate must bind asymmetrically in the active site of these enzymes. However, the coenzyme may have an altered orientation or active site environment in five of the mutant enzymes that display less intense ellipticity bands. The mutant enzyme in which lysine 87 is replaced by threonine has very weak ellipticity at 400 nm. Since lysine 87 forms a Schiff base with pyridoxal phosphate in the wild type enzyme, our results demonstrate the importance of the Schiff base linkage for rigid or asymmetric binding. Although the mutant enzymes display spectra in the presence of L-serine that differ from that of the wild type enzyme, addition of alpha-glycerol 3-phosphate converts the spectra of two of the mutant enzymes to that of the wild type enzyme. We conclude that this alpha-subunit ligand may produce a conformational change in the alpha-subunit that is transmitted to the mutant beta-subunits and partially corrects conformational alterations in the mutant enzymes.