The regulatory properties of yeast pyruvate kinase.

The kinetics of pyruvate kinase from Saccharomyces cerevisiae were studied in assays at pH 6.2 where the relationships between the initial velocities of the catalysed reaction and the concentrations of the substrates ADP, phosphoenolpyruvate and Mg2+ are non-hyperbolic. The findings were represented empirically by the exponential model for a regulatory enzyme. The analysis shows that ADP, phosphoenolpyruvate and Mg2+ display positive homotropic interaction in their binding behaviour with (calculated) Hill slopes at half-saturation equal to 1.06, 2.35 and 3.11 respectively [Ainsworth (1977) J. Theor. Biol. 68, 391-413]. The direct heterotropic interaction between ADP and phosphoenolpyruvate is small and negative, but the overall interaction between these substrates becomes positive when their positive interactions with Mg2+ are taken into account. The heterotropic interactions of the substrates, though smaller in magnitude, are comparable with those revealed by the rabbit muscle enzyme [Ainsworth, Kinderlerer & Gregory (1983) Biochem. J. 209, 401-411], and it is suggested that they have a common origin in charge interactions within the active site.     

The regulatory properties of yeast pyruvate kinase. Effect of pH.

The kinetics of pyruvate kinase from Saccharomyces cerevisiae were studied at 25 degrees C as a function of the concentrations of the substrates ADP, phosphoenolpyruvate and Mg2+ and the effector H+ in the pH range 5-6.6. The enzyme was activated by 100 mM-K+ and 32 mM-NH4+ throughout. It was found that the data could be described by the exponential model for a regulatory enzyme. On that basis, it was concluded that the binding of H+ is positively interactive and that the protonated enzyme is catalytically inactive. It was also found that H+ interacts positively with phosphoenolpyruvate but negatively with both ADP and Mg2+.     

The regulatory properties of rabbit muscle pyruvate kinase. The influence of substrate concentrations

The kinetics of rabbit muscle pyruvate kinase were studied in assays at pH 7.4, where the relationships between the initial velocities of the catalysed reaction and the concentrations of substrates ADP, phosphoenolpyruvate and Mg2+ are non-hyperbolic. The data were used to test the applicability of the exponential model for a regulatory enzyme, which has been here extended to describe the behaviour of a three-substrate enzyme. It appears that the data can be represented by the model and as a result permit the conclusion that the substrates influence one another's binding by the same type of charge interactions that are evident in the Michaelis-Menten kinetics of the enzyme observed at pH 6.2. Evidence is also presented indicating that MgADP acts as a dead-end inhibitor of the enzyme at pH 7.4.     

The regulatory properties of rabbit muscle pyruvate kinase. The influence of effector concentrations

The initial velocity of the reaction catalysed by rabbit muscle pyruvate kinase was studied as a function of the concentrations of the modifiers phenylalanine and fructose 1,6-bisphosphate under conditions where the relationships between the initial velocities and the concentrations of substrates are non-hyperbolic. It is shown that these data can be represented by the exponential model for a regulatory enzyme.     

The preparation and properties of pyruvate kinase from yeast

A new method is described for the preparation of pyruvate kinase from yeast. This eliminates proteolysis during the preparation. The molecular weight of yeast pyruvate kinase is 215000, and it is composed of four subunits. Such properties of the enzyme as its extinction coefficient, cold-lability, thiol-group reactivity and binding of Mn(2+) ions are compared with those previously reported for yeast pyruvate kinase prepared by different methods. The specific activity is significantly higher than previously observed, but otherwise the enzyme is similar, apart from its molecular weight and Mn(2+)-binding characteristics, to preparations from Saccharomyces cerevisiae obtained in this laboratory (e.g. Fell et al., 1972, and references therein) and that of C. H. Suelter (e.g. Kuczenski & Suelter, 1971, and references therein), and is different from the enzyme isolated from Saccharomyces carlsbergensis by B. Hess and his co-workers (e.g. Wieker & Hess, 1972, and references therein).     

The regulatory properties of rabbit muscle pyruvate kinase. The effect of pH.

The regulatory behavior of rabbit pyruvate kinase has been studied as a function of pH. The initial velocity of the enzyme-catalysed reaction as a function of ADP concentration was analysed with the exponential model for a regulatory enzyme. The analysis of the exponential model parameters as functions of pH provided pK values of 6.6 and 8.08 for the free enzyme in its fully ADP-bound conformation. By contrast, the binding of ADP to the ADP-free conformation of the free enzyme did not involve groups that ionize within the pH range (6.2-8.5) of these experiments. The results suggest that homotropic allosteric interactions actually alter the mode of ADP binding. The pK values of 6.63 and 9.00 determined from the analysis of V as a function of pH are readily interpreted in terms of a direct phosphoryl-transfer mechanism in which the beta-phosphoryl group of ADP (pK 6.63) acts as the nucleophile and a lysine epsilon-amino group (pK 9.0) acts as the proton donor in the pyruvate kinase reaction.     

Purification and regulatory properties of pyruvate kinase from Veillonella parvula.

The nonglycolytic, anaerobic organism Veillonella parvula M4 has been shown to contain an active pyruvate kinase. The enzyme was purified 126-fold and was shown by disc-gel electrophoresis to contain only two faint contaminating bands. The purified enzyme had a pH optimum of 7.0 in the forward direction and exhibited sigmoidal kinetics at varying concentrations o-f phosphoenol pyruvate (PEP), adenosine 5'-monophosphate (AMP), and Mg-2+ ions with S0.5 values of 1.5, 2.0, and 2.4 mM, respectively. Substrate inhibition was observed above 4 m PEP. Hill plots gave slope values (n) of 4.4 (PEP), 2.8 (adenosine 5'-diphosphate), and 2.0 (Mg-2+), indicating a high degree of cooperativity. The enzyme was inhibited non-competitively by adenosine 5'-triphosphate (Ki = 3.4 mM), and this inhibition was only slightly affected by increasing concentration of Mg-2+ ions to 30 mM. Competitive inhibition was observed with 3-phosphoglycerate, malate, and 2,3-diphosphoglycerate but only at higher inhibitor concentrations. The enzyme was activated by glucose-6-phosphate (P), fructose-6-P, fructose-1,6-diphosphate (P2), dihydroxyacetone-P, and AMP; the Hill coefficients were 2.2, 1.8, 1.5, 2.1, and 2.0, respectively. The presence of each these metabolites caused substrate velocity curves to change from sigmoidal to hyperbolic curves, and each was accompanied by an increase in the maximum activity, e.g., AMP greater than fructose-1,6-P2 greater than dihydroxyacetone-P greater than glucose-6-P greater than fructose-6-P. The activation constants for fructose-1,6-P2, AMP, and glucose-6-P were 0.3, 1.1, and 5.3 mM, respectively. The effect of 5 mM fructose-1,6-P2 was significantly different from the other compounds in that this metabolite was inhibitory between 1.2 and 3 mM PEP. Above this concentration, fructose-1,6-P2 activated the enzyme and abolished substrate inhibition by PEP. The enzyme was not affected by glucose, glyceraldehyde-3-P, 2-phosphoglycerate, lactate, malate, fumerate, succinate, and cyclic AMP. The results suggest that the pyruvate kinase from V. parvula M4 plays a central role in the control of gluconeogenesis in this organism by regulating the concentration of PEP.     

The characterization and regulatory properties of pyruvate kinase from cotton seeds

Pyruvate kinase (ATP: pyruvate phosphotransferase, EC 2.7.1.40) was partially purified from cotton seeds. The enzyme shows normal kinetics toward phosphoenol-pyruvate, ADP, and magnesium or manganese. Of nearly 50 metabolites tested, the enzyme is inhibited only by ATP, UTP, citrate, and malate, and activated by AMP, GMP, and fumarate. The inhibition by citrate and ATP is not due to metal chelation; both compounds appear to directly affect the enzyme. The kinetics of the activations by AMP and by fumarate suggest the existence of separate activator sites for the two compounds.It is suggested that cotton seed pyruvate kinase is a regulatory enzyme, although it differs markedly from the regulatory pyruvate kinases which have been described in animals and in microorganisms. This is the first instance in which regulatory properties have been reported for a pyruvate kinase from a higher plant.     

Purification and regulatory properties of pigeon erythrocyte pyruvate kinase

1. Pigeon erythrocyte pyruvate kinase (PK) was purified 22,000 fold by successive column chromatography on Sephadex DEAE A-50 and Red Agarose. The resulting enzyme preparation had a specific activity of 815.3 U/mg protein and an overall yield of 18.5%. 2. The molecular weight, as determined by gel filtration on Sephadex G-200 was 152,000. 3. Isoelectric focusing in the pH range of 3-10 showed that pigeon erythrocyte contained at least 3 PK isozymes with isoelectric points of 5, 5.7 and 6. 4. The variation of activity of PK at various ADP and phosphoenolpyruvate (PEP) concentrations was studied. The Km values for ADP and PEP were 0.40 and 0.46 mM respectively. 5. The enzyme was activated by FDP, and inhibited by ATP, highly phosphorylated inositol derivatives and 2,3-DPG: 6. It was activated by K+ and Mg2+ ions. 7. Phosphorylated hexoses and Pi stimulated the activity of PK. 8. The regulatory role of PK of pigeon erythrocytes, which lack the typical 2,3-DPG bypass, is discussed.     

Spinach pyruvate kinase isoforms : partial purification and regulatory properties

Pyruvate kinase from spinach (Spinacea oleracea L.) leaves consists of two isoforms, separable by blue agarose chromatography. Both isoforms share similar pH profiles and substrate and alternate nucleotide K_m values. In addition, both isoforms are inhibited by oxalate and ATP and activated by AMP. The isoforms differ in their response to three key metabolites; citrate, aspartate, and glutamate. The first isoform is similar to previously reported plant pyruvate kinases in its sensitivity to citrate inhibition. The K_i for this inhibition is 1.2 millimolar citrate. The second isoform is not affected by citrate but is regulated by aspartate and glutamate. Aspartate is an activator with a K_a of 0.05 millimolar, and glutamate is an inhibitor with a K_i of 0.68 millimolar. A pyruvate kinase with these properties has not been previously reported. Based on these considerations, we suggest that the activity of the first isoform is regulated by respiratory metabolism. The second isoform, in contrast, may be regulated by the demand for carbon skeletons for use in ammonia assimilation.     

Secondary kinase reactions catalyzed by yeast pyruvate kinase.

1. Yeast pyruvate kinase (EC 2.7.1.40) catalyzes, in addition to the primary, physiologically important reaction, three secondary kinase reactions, the ATP-dependent phosphorylations of fluoride (fluorokinase), hydroxylamine (hydroxylamine kinase) and glycolate (glycolate kinase). 2. These reactions are accelerated by fructose-1,6-bisphosphate, the allosteric activator of the primary reaction. Wth Mg2+ as the required divalent cation, none of these reactions are observed in the absence of fructose-biphosphate. With Mn2+, fructose-bisphosphate is required for the glycolate kinase reaction, but merely stimulates the other reactions. 3. The effect of other divalent cations and pH on three secondary kinase reactions was also examined. 4. Results are compared with those obtained from muscle pyruvate kinase and the implications of the results for the mechanism of the yeast enzyme are discussed.     

The proton transfer reactions catalyzed by yeast pyruvate kinase.

1. The proton-transfer reactions of yeast pyruvate kinase (EC 2.7.1.40) were studied. Proton-transfer from C-3 of phosphoenolpyruvate to water occurs only in the presence of the phosphoryl-acceptor ADP. Proton transfer from C-3 of pyruvate to water occurs only in the presence of ATP. However, the proton transfer in the latter case occurs 10-100 times faster than phosphoryl transfer; this supports a mechanism in which proton transfer precedes phosphoryl transfer in the reverse reaction of pyruvate kinase. 2. The characteristics of proton-transfer reactions of yeast pyruvate kinase were compared with those previously reported for rabbit muscle pyruvate kinase (Robinson, JL. and Rose, I.A. (1972) J. Biol. Chem. 247, 1096-1105). The pH-profiles and the divalent cation dependencies were similar for Fru-1,6-P2-activated yeast pyruvate kinase and the muscle enzyme. Pyruvate enolization by yeast pyruvate kinase has an absolute requirement for ATP in contrast to enolization by the muscle enzyme which proceeds when ATP is replaced by Pi or other dianions. 3. Fructose-1,6-bisphosphate was shown to affect the catelytic steps of yeast pyruvate kinase in addition to the binding of substrates. Its role depends on the divalent cation used to activate the enzyme.     

Selectivity of pyruvate kinase for Na+ and K+ in water/dimethylsulfoxide mixtures.

In aqueous media, muscle pyruvate kinase is highly selective for K+ over Na+. We now studied the selectivity of pyruvate kinase in water/dimethylsulfoxide mixtures by measuring the activation and inhibition constants of K+ and Na+, i.e. their binding to the monovalent and divalent cation binding sites of pyruvate kinase, respectively [Melchoir J.B. (1965) Biochemistry 4, 1518-1525]. In 40% dimethylsulfoxide the K0.5 app for K+ and Na+ were 190 and 64-fold lower than in water. Ki app for K+ and Na+ decreased 116 and 135-fold between 20 and 40% dimethylsulfoxide. The ratios of Ki app/K0.5 app for K+ and Na+ were 34-3.5 and 3.3-0.2, respectively. Therefore, dimethylsulfoxide favored the partition of K+ and Na+ into the monovalent and divalent cation binding sites of the enzyme. The kinetics of the enzyme at subsaturating concentrations of activators show that K+ and Mg2+ exhibit high selectivity for their respective cation binding sites, whereas when Na+ substitutes K+, Na+ and Mg2+ bind with high affinity to their incorrect sites. This is evident by the ratio of the affinities of Mg2+ and K+ for the monovalent cation binding site, which is close to 200. For Na+ and Mg2+ this ratio is approximately 20. Therefore, the data suggest that K+ induces conformational changes that prevent the binding of Mg2+ to the monovalent cation binding site. Circular dichroism spectra of the enzyme and the magnitude of the transfer and apparent binding energies of K+ and Na+ indicate that structural arrangements of the enzyme induced by dimethylsulfoxide determine the affinities of pyruvate kinase for K+ and Na+.     

The effects of aestivation on the catalytic and regulatory properties of pyruvate kinase from Helix aspersa.

1. Pyruvate kinase was partially purified from the foot, mantle, and digestive gland of active and aestivating snails. 2. At pH 7.0 the apparent Km values for phosphoenolpyruvate (PEP) were 0.064 mmol/l for the enzyme from foot and 0.071 mmol/l for the enzyme from mantle; those for ADP were 0.35 mmol/l for the foot enzyme and 0.33 mmol/l for the mantle enzyme. 3. Both enzymes were inhibited by alanine, and this could be reversed by fructose 1,6-bisphosphate (FBP), although FBP alone was a weak activator. 4. Decreasing the pH to 6.5 markedly increased the inhibition by alanine and reduced the response to FBP. 5. The enzymes from these tissues of aestivating snails showed a small decrease in their affinity for PEP and a small increase in the effectiveness of alanine as an inhibitor. 6. These changes are indicative of a down-regulation of this enzyme which is consistent with the observations in other species during metabolic depression. 7. In contrast the enzyme from the digestive gland of active animals showed sigmoidal saturation kinetics for PEP with a S0.5 of 1.2 mmol/l, but had a markedly higher affinity for PEP, S0.5 = 0.20 mmol/l during aestivation. This may be indicative of other metabolic changes occurring in the digestive gland.     

Purification, catalytic and regulatory properties of Rana ridibunda erythrocyte pyruvate kinase

Pyruvate kinase of Rana ridibunda erythrocytes is one of the regulatory enzymes of glycolysis. PK was purified about 7800-fold. The purified enzyme showed on SDS-electrophoresis three protein bands with an apparent molecular weight of between 60 and 65 kD. The enzyme is subject to activation by FDP and to inhibition by ATP. It showed Km values for PEP and ADP of 0.095 and 0.98 mM respectively. It was activated by K+, Mg2+ and Ca2+ ions whereas it was inhibited by Na+ ions. The role of PK of Rana ridibunda erythrocytes, as a key and rate controlling enzyme of the glycolytic flux is discussed.     

NH4+对L-色氨酸发酵的影响

目的:探究NH₄⁺浓度对大肠杆菌E.coli TRTH发酵生产L-色氨酸的影响。方法:通过外源添加试验,利用30 L发酵罐进行分批补料发酵试验,考察E. coli TRTH发酵生产L-色氨酸过程中生物量、L-色氨酸产量、有机酸含量、耗糖速率、发酵液中NH₄⁺浓度及质粒稳定性变化。建立了大肠杆菌合成L-色氨酸的代谢流平衡模型,应用 MATLAB 软件计算出E. coli TRTH发酵中后期代谢网络的代谢流分布。结果:发酵结果显示,利用NaOH和氨水混合补料,控制NH₄⁺浓度在120 mmol/L以下,菌体能够以较长时间和较高比生长速率保持对数生长,最终菌体生物量和L-色氨酸产量分别提高了12.16%和19.80%。随着NH₄⁺浓度的增加,发酵液中丙酮酸、乳酸及乙酸浓度均略有增加,细胞质粒稳定性下降。控制NH₄⁺浓度在120 mmol/L以下,E. coli TRTH发酵生产L-色氨酸的代谢流量分析结果表明,EMP途径的代谢流量降低7.31%,PP途径的代谢流量增加7.14%,TCA循环的代谢流量降低22.04%。结论:高浓度的NH₄⁺导致菌体生长提前结束,耗糖速率降低,产酸受阻,控制NH₄⁺浓度在120 mmol/L以下,解除了NH₄⁺对菌体生长和产物生成的抑制,使得菌体生物量和L-色氨酸产量大幅提高,实现了高密度发酵培养的目的。