Allosteric pyruvate kinase from Phycomyces blakesleeanus: physicochemical and regulatory properties

1988 ◽  
Vol 66 (2) ◽  
pp. 148-157 ◽  
Author(s):  
Félix Busto ◽  
Pilar Del Valle ◽  
Joaquín Soler
Keyword(s):  
Molecular Weight ◽  
Pyruvate Kinase ◽  
Specific Activity ◽  
Substrate Inhibition ◽  
Kinetic Properties ◽  
Saturation Curve ◽  
Phycomyces Blakesleeanus ◽  
Competitive Inhibitors ◽  
Fructose 1,6 Bisphosphate ◽  
Regulatory Properties

Pyruvate kinase from mycelium of Phycomyces blakesleeanus NRRL 1555 (−) was purified approximately 500-fold to a final specific activity of 25 U∙mg protein−1. The native enzyme has a molecular weight of 230 000. The enzyme appeared to be a tetramer with apparently identical subunits of 58 000 each. The enzyme from Phycomyces requires Mg2+ for activity, but not K+ or NH4. It showed a transition temperature at 36 °C. L-Alanine and ATP allosterically inhibited the enzyme by increasing the positive homotropic interactions for phosphoenolpyruvate and abolishing them for Mg2+ ions. Both effectors appeared to be competitive inhibitors with regard to ADP. Fructose 1,6-bisphosphate activates the Phycomyces pyruvate kinase allosterically by transforming the sigmoidal saturation curves to a hyperboolic form for phosphoenolpyruvate and Mg2+. Furthermore, fructose 1,6-bisphosphate relieved the inhibition caused by ATP and L-alanine. A lowering of the pH for reaction also activates the enzyme by abolishing the sigmoidal saturation curve for phosphoenolpyruvate, but produces substrate inhibition. The kinetic properties of Phycomyces pyruvate kinase are compatible to that of an allosteric K-type enzyme.

scholarly journals Kinetic studies on the regulation of rabbit liver pyruvate kinase

1973 ◽  
Vol 131 (2) ◽  
pp. 287-301 ◽  
Author(s):  
M. G. Irving ◽  
J. F. Williams
Keyword(s):  
Pyruvate Kinase ◽  
Specific Activity ◽  
Deae Cellulose ◽  
Kinetic Studies ◽  
Rabbit Liver ◽  
Hill Coefficient ◽  
Saturation Curve ◽  
Low Concentrations ◽  
Ammonium Sulphate Fractionation ◽  
The Hill

Two kinetically distinct forms of pyruvate kinase (EC 2.7.1.40) were isolated from rabbit liver by using differential ammonium sulphate fractionation. The L or liver form, which is allosterically activated by fructose 1,6-diphosphate, was partially purified by DEAE-cellulose chromatography to give a maximum specific activity of 20 units/mg. The L form was allosterically activated by K+ and optimum activity was recorded with 30mm-K+, 4mm-MgADP-, with a MgADP-/ADP2- ratio of 50:1, but inhibition occurred with K+ concentrations in excess of 60mm. No inhibition occurred with either ATP or GTP when excess of Mg2+ was added to counteract chelation by these ligands. Alanine (2.5mm) caused 50% inhibition at low concentrations of phosphoenolpyruvate (0.15mm). The homotropic effector, phosphoenolpyruvate, exhibited a complex allosteric pattern (nH+2.5), and negative co-operative interactions were observed in the presence of low concentrations of this substrate. The degree of this co-operative interaction was pH-dependent, with the Hill coefficient increasing from 1.1 to 3.2 as the pH was raised from 6.5 to 8.0. Fructose 1,6-diphosphate interfered with the activation by univalent ions, markedly decreased the apparent Km for phosphoenolpyruvate from 1.2mm to 0.2mm, and transformed the phosphoenolpyruvate saturation curve into a hyperbola. Concentrations of fructose 1,6-diphosphate in excess of 0.5mm inhibited this stimulated reaction. The M or muscle-type form of the enzyme was not activated by fructose 1,6-diphosphate and gave a maximum specific activity of 0.3 unit/mg. A Michaelis–Menten response was obtained when phosphoenolpyruvate was the variable substrate (Km+0.125mm), and this form was inhibited by ATP, as well as alanine, even in the presence of excess of Mg2+.

scholarly journals Partial purification of topoisomerase IB from serum of diabetic patients and study it's kinetic properties and molecular weight

2019 ◽  
Vol 23 (10) ◽  
pp. 46
Author(s):  
Saif M. Hasan ◽  
Firas T. Maher ◽  
Nagham Q. Kadhim
Keyword(s):  
Molecular Weight ◽  
Specific Activity ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Maximum Velocity ◽  
Partial Purification ◽  
Diabetic Patients ◽  
Kinetic Properties ◽  
Topoisomerase Ib ◽  
Electrophoresis Method

This study was done to partially purification of  topoisomerase IB from serum of diabetic patients using Gel filtration technique, by using Sephadex G 100 gel. A single peak in fraction four has been obtained, and the degree of purification (17.1) fold, enzyme yield (108.2%) and specific activity (0.189ng/mg). Kinetics studies for the partial purified enzyme were carried out which showed optimal concentration of  substrate which was (0.1ng/ml), Michael's - Menten constant (Km=0.033ng) and maximum velocity (Vmax=0.90 ng/ml), while optimum Temperature was (37C°) and optimum pH was (7.5). The molecular weight of the partial purified enzyme has been determined by gel electrophoresis method, in presence of polyacrylamide  gel and sodium dodecyl sulphate (SDS-PAGE) which showed that the approximated molecular weight was (66KD).   http://dx.doi.org/10.25130/tjps.23.2018.168 

scholarly journals The biosynthesis of granulose by Clostridium pasteurianum

1974 ◽  
Vol 144 (3) ◽  
pp. 503-511 ◽  
Author(s):  
R L Robson ◽  
R M Robson ◽  
J G Morris
Keyword(s):  
Specific Activity ◽  
Competitive Inhibitor ◽  
Partial Purification ◽  
Clostridium Pasteurianum ◽  
Saturation Curve ◽  
Wild Type ◽  
Competitive Inhibitors ◽  
Fine Control ◽  
Adp Glucose Pyrophosphorylase ◽  
High Concentrations

1. Mutant strains of Clostridium pasteurianum were obtained, which are unable to synthesize granulose (an intracellularly accumulated amylopectin-like α-polyglucan). 2. These mutants lacked either (a) ADP-glucose pyrophosphorylase (EC 2.7.7.27), or (b) granulose synthase (i.e. ADP-glucose–α-1,4-glucan glucosyltransferase, EC 2.4.1.21). 3. Although both of these enzymes were constitutively synthesized by the wild-type organism, massive deposition of granulose in a sporulating culture coincided with a threefold increase in the specific activity of ADP-glucose pyrophosphorylase. 4. The soluble ADP-glucose pyrophosphorylase was partially purified (33-fold). Its ATP-saturation curve was not sigmoidal and its activity was not enhanced by phosphorylated intermediates of glycolysis, pyruvate, NAD(P)H or pyridoxal 5′-phosphate. ADP at relatively high concentrations acted as a competitive inhibitor (Ki=19mm). 5. The dependence of granulose synthase on a suitable polyglucan primer was demonstrated by using enzyme obtained from a granulose-free mutant strain (lacking ADP-glucose pyrophosphorylase). 6. Partial purification of granulose synthase from wild-type strains was facilitated by its being bound to the native particles of granulose. No activator was discovered, but ADP, AMP and pyridoxal 5′-phosphate were competitive inhibitors, ADP being most effective (Ki about 0.2mm). 7. It would appear that the synthesis of granulose in Cl. pasteurianum is not subject to the positive, fine control that is a feature of glycogen biosynthesis in most bacteria.

The Kinetics Of The Inhibition Of Human Plasma Kallikrein By Plasma Protease Inhibitors: Role Of High Molecular Weight Kininogen

1981 ◽  
Author(s):  
M Schapira ◽  
A James ◽  
C F Scott ◽  
F Kueppers ◽  
H L James ◽  
...  
Keyword(s):  
Molecular Weight ◽  
High Molecular Weight ◽  
Protease Inhibitors ◽  
Substrate Inhibition ◽  
Catalytic Efficiency ◽  
Reaction Rates ◽  
Plasma Kallikrein ◽  
High Molecular Weight Kininogen ◽  
Α2 Macroglobulin ◽  
Competitive Inhibitors

Plasma kallikrein (KAL) is inhibited by several plasma protease inhibitors, including C1-inhibitor (C1-INH), antithrombin III (ATIII), α1-antitrypsin (α1AT), and α2-macroglobulin (α2M). To assess the mechanism of action and the relative importance of these inhibitors, we have undertaken inhibition studies with purified proteins, using H-D-Pro- Phe-Arg-Nan as KAL substrate. Inhibition was competitive with C1INH, ATIII, and α1AT and noncompetitive with α2M. KAL retained 14% of its catalytic efficiency when complexed to α2M. The rate constants for inhibition by C1INH, ATIII, α1AT, and α2M were 28, 0.18, 0.003, and 6.9 M-ls-1(10-3) respectively. Michaelis-Menten kinetics was observed for the inhibition by ATIII, αlAT, and α2M. The constants for the rate-limiting formation of the irreversible complexes were 16, 0.27 and 2.0 s-1(xl02), while the KI’s for the reversible complex were 86, 63, and 0.29 γM, respectively for ATIII, α1AT and α2M. In_contrast, no Michaelis-Menten complex was observed when C1INH inhibited KAL. These results indicate that (a) C1INH is the most efficient inhibitor of KAL, (b) α2M is a significant inhibitor of KAL, (c) both ATIII and αlAT are probably not significant inhibitors of KAL. We have shown that high molecular weight kininogen (HMWK) decreases the inactivation rate of KAL by C1INH by forming a reversible complex with KAL. We now report that the reaction rates of KAL with ATIII and α1AT, which are competitive inhibitors, were decreased by 50%, when HMWK was 1 U/ml or 0.73 γM. When KAL was inhibited by α2M, a noncompetitive inhibitor, the inactivation rates were identical in the presence or absence of HMWK. Since HMWK protects KAL from being inhibited by competitive inhibitors but not by a noncompetitive one, these results confirm our previous observation indicating that the binding site for IMWK on KAL is closely linked to its catalytic site.

Pyruvate kinase from the moderate halophile, Vibrio costicola

1977 ◽  
Vol 55 (8) ◽  
pp. 825-833 ◽  
Author(s):  
Eveline de Médicis ◽  
Bertrand Rossignol
Keyword(s):  
Molecular Weight ◽  
Potassium Chloride ◽  
Pyruvate Kinase ◽  
Specific Activity ◽  
Chloride Concentration ◽  
Maximum Velocity ◽  
Maximal Activity ◽  
Maximum Activity ◽  
Moderate Halophile ◽  
Exclusion Chromatography

Pyruvate kinase (EC 2.7.1.40)from Vibrio costicola was purified to homogeneity. The tetramer had a molecular weight of 270 000 measured by exclusion chromatography on Sepharose 4B and was dissociated into apparently identical subunits with a molecular weight of 65 000 measured by SDS–polyacrylamide gel.The kinetic parameters Vm, K0.5, and n were measured at variable KCl concentrations. Both affinity and activity were inhibited when potassium chloride concentration was at 0.025 M, a fact which indicates that low potassium concentration is required for activity. Maximal activity and affinity were obtained at 0.125 M KCl. Potassium chloride appeared as an allosteric inhibitor in the range of 0.125 M to 1.025 M. Above 1.025 M KCl, both maximum velocity and affinity were decreased. NaCl had the same effect as KCl. The pH profiles of K0.5 and Vm of the phosphoenolpyruvate dependence revealed two main pK values: at 6.4 which controls affinity, and at 8.6, which controls activity.The activity of the enzyme showed a hysteretic response to salt. The specific activity measured under fixed standard conditions of temperature and salt concentrations showed a reversible variation which depended upon the preincubation conditions of temperature and salt concentration. The maximum activity tested was reached after low (0.1 M) or high (2 M) KCl preincubation at room temperature.

scholarly journals Purification and properties of 6-phosphogluconate dehydrogenase from rabbit mammary gland

1975 ◽  
Vol 151 (2) ◽  
pp. 263-270 ◽  
Author(s):  
S A Betts ◽  
R J Mayer
Keyword(s):  
Molecular Weight ◽  
Mammary Gland ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Phosphogluconate Dehydrogenase ◽  
Purification And Properties ◽  
Competitive Inhibitors ◽  
Final Purification Step

1. 6-Phosphogluconate dehydrogenase from rabbit mammary gland was purified to homogeneity by the criterion of polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate. The molecular weight of the subunit is 52 000. The enzyme was purified 150-fold with a final specific activity of 20 mumol of NADP+ reduced/min per mg of protein and overall yield of 3%. The molecular weight of the native enzyme is estimated to be 104 000 from gel-filtration studies. The final purification step was carried out by affinity chromatography with NADP+-Sepharose. 2. The Km values for 6-phosphogluconate and NADP+ are approx. 54 muM and 23 muM respectively. 3. Citrate and pyrophosphate are competitive inhibitors of the enzyme with respect to both 6-phosphogluconate and NADP+. 4. MgCl2 affects the apparent Km for NADP+ at saturating concentrations of 6-phosphogluconate.

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

1983 ◽  
Vol 209 (2) ◽  
pp. 413-415 ◽  
Author(s):  
R B Gregory ◽  
S Ainsworth ◽  
J Kinderlerer
Keyword(s):  
Pyruvate Kinase ◽  
Initial Velocity ◽  
Exponential Model ◽  
Rabbit Muscle ◽  
Muscle Pyruvate Kinase ◽  
Fructose 1,6 Bisphosphate ◽  
Regulatory Properties

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.

Purification and Properties of Phosphoenolpyruvate Carboxykinase from C4 Plants

1986 ◽  
Vol 13 (5) ◽  
pp. 577 ◽  
Author(s):  
JN Burnell
Keyword(s):  
Molecular Weight ◽  
Phosphoenolpyruvate Carboxykinase ◽  
C4 Plants ◽  
Kinetic Properties ◽  
Panicum Maximum ◽  
Ph Optimum ◽  
Wide Ph Range ◽  
Pep Carboxykinase ◽  
Nucleotide Specificity ◽  
Fructose 1,6 Bisphosphate

Phosphoenolpyruvate (PEP) carboxykinase (EC 4.1.1.49) from the leaves of Urochloa panicoides, Chloris gayana and Panicum maximum has been purified to homogeneity and its properties determined. The enzyme from all three PEP carboxykinase-type C4 plants have similar physical and kinetic properties. The native enzyme has a molecular weight of 380 000 and its monomeric molecular weight is about 64 000, suggesting the enzyme is hexameric. It is active over a wide pH range and has a pH optimum between 7.4 and 8.2, has a wide nucleotide specificity, has an absolute requirement for Mn2+ and is stimulated by C-. The enzyme is inhibited by 3-phosphoglyceric acid, fructose 6-phosphate and fructose 1,6-bisphosphate; the mechanism of inhibition is discussed. The purified PEP carboxykinase is unable to catalyse the conversion of oxaloacetate to pyruvate, nor does it possess pyruvate kinase activity. These findings are discussed in relation to the C4 photosynthetic pathway operating in PEP carboxykinase-type C4 plants.

scholarly journals The preparation and properties of pyruvate kinase from yeast

1974 ◽  
Vol 139 (3) ◽  
pp. 665-675 ◽  
Author(s):  
David A. Fell ◽  
Peter F. Liddle ◽  
Arthur R. Peacocke ◽  
Raymond A. Dwek
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Molecular Weight ◽  
Pyruvate Kinase ◽  
Extinction Coefficient ◽  
Specific Activity ◽  
Thiol Group ◽  
New Method ◽  
Saccharomyces Carlsbergensis ◽  
Binding Characteristics

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 Mn2+ 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 Mn2+-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).

Purification and properties of phosphoenolpyruvate carboxylase from green leaves of maize

1979 ◽  
Vol 44 (6) ◽  
pp. 1835-1840 ◽  
Author(s):  
Jaroslav Mareš ◽  
Jana Barthová ◽  
Sylva Leblová
Keyword(s):  
Molecular Weight ◽  
Specific Activity ◽  
Deae Cellulose ◽  
Chloride Ions ◽  
Magnesium Ions ◽  
Ph Optimum ◽  
Green Leaves ◽  
Purification And Properties ◽  
Fructose 1,6 Bisphosphate ◽  
No Activation

Phosphoenolpyruvate carboxylate was isolated from green leaves of maize (Zea mays L.) by a procedure including fractionation with ammonium sulphate, chromatography on DEAE-cellulose and preparative electrophoresis on polyacrylamide gel. The specific activity of the electrophoretically homogeneous enzyme was 23 U/mg. Its molecular weight was about 405 000, pH optimum was within the range 7.9 to 8.3, Km for phosphoenolpyruvate was 1.05 . 10-3 and the apparent Km for the magnesium ions was 8.0 . 10-4M. The enzyme was inhibited by malate, aspartate, citrate, pyruvate, ATP and ADP and chloride ions. It was strongly activated by glycine and glucose 6-phosphate and to a lesser degree by glucose 1-phosphate and fructose 1,6-bisphosphate; no activation by orthophosphate and 3-phosphoglycerate was observed.

Sign in / Sign up

Export Citation Format

Share Document