On the Role of Magnesium in the Reaction of the Pyruvate Kinase from Salmonella typhimurium

Abstract The kinetics of the two purified forms of pyruvate kinase from Salmonella typhimurium LT-2 were studied in assays at pH 6.8 where the relationships between the initial velocities of the catalysed reactions and Mg2+ are non-hyperbolic. The analysis show that Mg2+ display positive homotropic interactions in their binding behaviour with Hill coefficient values of 2.5 and 1.2 for the form I and II, respectively. The binding sites of the cation to the pyruvate kinases seem to be independent to those for phosphoenolpyruvate and adenosine 5′-diphosphate; changes in the magnesium concentration might be of physiological significance in relation to a rapid regeneration of adenosine 5′-triphosphate by means of the pyruvate kinase reaction.

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Role of ArgR in Activation of the ast Operon, Encoding Enzymes of the Arginine Succinyltransferase Pathway in Salmonella typhimurium

Journal of Bacteriology ◽

10.1128/jb.181.6.1934-1938.1999 ◽

1999 ◽

Vol 181 (6) ◽

pp. 1934-1938 ◽

Cited By ~ 28

Author(s):

Chung-Dar Lu ◽

Ahmed T. Abdelal

Keyword(s):

Salmonella Typhimurium ◽

Binding Sites ◽

Regulatory Proteins ◽

Receptor Protein ◽

Carbon Limitation ◽

Consensus Sequences ◽

Flanking Region ◽

Starvation Conditions ◽

Combined Data

ABSTRACT The ast operon, encoding enzymes of the arginine succinyltransferase (AST) pathway, was cloned from Salmonella typhimurium, and the nucleotide sequence for the upstream flanking region was determined. The control region contains several regulatory consensus sequences, including binding sites for NtrC, cyclic AMP receptor protein (CRP), and ArgR. The results of DNase I footprintings and gel retardation experiments confirm binding of these regulatory proteins to the identified sites. Exogenous arginine induced AST under nitrogen-limiting conditions, and this induction was abolished in an argR derivative. AST was also induced under carbon starvation conditions; this induction required functional CRP as well as functional ArgR. The combined data are consistent with the hypothesis that binding of one or more ArgR molecules to a region between the upstream binding sites for NtrC and CRP and two putative promoters plays a pivotal role in modulating expression of theast operon in response to nitrogen or carbon limitation.

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The regulatory properties of yeast pyruvate kinase. Effect of fructose 1,6-bisphosphate

Biochemical Journal ◽

10.1042/bj2340691 ◽

1986 ◽

Vol 234 (3) ◽

pp. 691-698 ◽

Cited By ~ 14

Author(s):

C N Morris ◽

S Ainsworth ◽

J Kinderlerer

Keyword(s):

Saccharomyces Cerevisiae ◽

Pyruvate Kinase ◽

Substrate Concentration ◽

Exponential Model ◽

Fructose Bisphosphate ◽

Inflected Form ◽

Fructose 1,6 Bisphosphate ◽

Regulatory Properties ◽

Kinetics Of ◽

Binding Behaviour

The kinetics of pyruvate kinase from Saccharomyces cerevisiae were studied in assays at pH 6.2 at 25 degrees C as a function of the concentrations of the substrates ADP, phosphoenolpyruvate and Mg2+ and the concentration of the effector fructose 1,6-bisphosphate. The enzyme was activated by 100 mM-K+ and 32 mM-NH4+ throughout. It was found that an increase in the fructose bisphosphate concentration from 24 microM to 1.2 mM brings about a transition from a sigmoidal to a non-inflected form in the relationships v = f([phosphoenolpyruvate]) and v = f([Mg2+]) together with a large increase in the affinity of these substrates for the enzyme. The binding behaviour of ADP is barely affected by the same change in effector concentration. By contrast, increase in fructose bisphosphate concentration below 24 microM increases the affinity of the enzyme for all its substrates and the sigmoidicity of the corresponding velocity-substrate-concentration relationships. As a result of this change in behaviour it has been found impossible to represent all the data by the exponential model for a regulatory enzyme, and it is suggested (supported by comparisons with previous work) that the failure may reflect a secondary action of the effector upon the enzyme.

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The regulatory properties of yeast pyruvate kinase

Biochemical Journal ◽

10.1042/bj2170641 ◽

1984 ◽

Vol 217 (3) ◽

pp. 641-647 ◽

Cited By ~ 4

Author(s):

C N Morris ◽

S Ainsworth ◽

J Kinderlerer

Keyword(s):

Pyruvate Kinase ◽

Active Site ◽

Exponential Model ◽

Positive Interactions ◽

Rabbit Muscle ◽

Muscle Enzyme ◽

Catalysed Reaction ◽

Regulatory Properties ◽

Kinetics Of ◽

Binding Behaviour

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.

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Investigation of the mechanism by which glucose analogues cause translocation of glucokinase in hepatocytes: evidence for two glucose binding sites

Biochemical Journal ◽

10.1042/bj3460413 ◽

2000 ◽

Vol 346 (2) ◽

pp. 413-421 ◽

Cited By ~ 21

Author(s):

Loranne AGIUS ◽

Mark STUBBS

Keyword(s):

Binding Site ◽

Binding Sites ◽

The Other ◽

Hill Coefficient ◽

Site Model ◽

Glucose Binding ◽

Glucose Analogues ◽

Hill Coefficients ◽

Effect Of Glucose ◽

Kinetics Of

Glucokinase translocates between the cytoplasm and nucleus of hepatocytes where it is bound to a 68 kDa protein. The mechanism by which glucose induces translocation of glucokinase from the nucleus was investigated using glucose analogues that are not phosphorylated by glucokinase. There was strong synergism on glucokinase translocation between effects of glucose analogues (glucosamine, 5-thioglucose, mannoheptulose) and sorbitol, a precursor of fructose 1-phosphate. In the absence of glucose or glucose analogues, sorbitol had a smaller effect than glucose on translocation. However, sorbitol potentiated the effects of glucose analogues. In the absence of sorbitol the effect of glucose on glucokinase translocation is sigmoidal with a Hill coefficient of 1.9 suggesting involvement of two glucose-binding sites. The effects of glucosamine and 5-thioglucose were also sigmoidal but with lower Hill Coefficients. In the presence of sorbitol, the effects of glucose, glucosamine and 5-thioglucose were hyperbolic. Mannoheptulose, unlike the other glucose analogues, had a hyperbolic effect on glucokinase translocation in the absence of sorbitol suggesting interaction with one site and was synergistic rather than competitive with glucose. The results favour a two-site model for glucokinase translocation involving either two glucose-binding sites or one binding-site for glucose and one for fructose 1-phosphate. The glucose analogues differed in their effects on the kinetics of purified glucokinase. Mannoheptulose caused the greatest decrease in co-operativity of glucokinase for glucose whereas N-acetylglucosamine had the smallest effect. The anomalous effects of mannoheptulose on glucokinase translocation and on the kinetics of purified glucokinase could be explained by a second glucose-binding site on glucokinase.

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Effects of secondary binding by activator and inhibitor peptides on covalent intermediates of pig pepsin

Biochemical Journal ◽

10.1042/bj1530701 ◽

1976 ◽

Vol 153 (3) ◽

pp. 701-712 ◽

Cited By ~ 15

Author(s):

T T Wang ◽

T Hofmann

Keyword(s):

Binding Sites ◽

Competitive Inhibition ◽

Catalytic Efficiency ◽

Acyl Transfer ◽

High Substrate Concentration ◽

Substrate Activation ◽

Substrate Cleavage ◽

Kinetics Of ◽

Hydrolysis Of

A number of peptides were found to increase the activity of pig pepsin towards small synthetic substrates. The activators increase transpeptidation of both the acyl-transfer and the amino-transfer types by as much as 45-fold. The effect on hydrolysis varies from inhibition to modest activation, but is always less than the effect on transpeptidation. The kinetics of substrate cleavage are the converse of non-competitive inhibition and show an increase in kcat. and no effect on Km values. Lineweaver-Burk plots of results obtained in the presence of the activators indicate a substrate activation at high substrate concentration. This appears to be a co-operative effect, since it is not observed in the absence of the activators. The activation is greatest at pH 4.7, less at pH 3.4, and at pH 2.0 is observable only with some of the activator peptides. The results show directly the effect of secondary binding on the catalytic efficiency of pepsin. The most effective activators are those that are most hydrophobic. The results suggest that binding in the secondary binding sites causes an increase in hydrophobicity in the catalytic site which results in increased stability of the acyl and amino intermediates, and preferential reaction with acceptors other than water. The implication that the present results strengthen the case for a role of covalent intermediates in the hydrolysis of good substrates (high kcat. values) is discussed.

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Kinetics of hexokinase D (‘glucokinase’) with inosine triphosphate as phosphate donor. Loss of kinetic co-operativity with respect to glucose

Biochemical Journal ◽

10.1042/bj2450625 ◽

1987 ◽

Vol 245 (3) ◽

pp. 625-629 ◽

Cited By ~ 7

Author(s):

D Pollard-Knight ◽

A Cornish-Bowden

Keyword(s):

High Glucose ◽

Kinetic Models ◽

Binding Sites ◽

Ternary Complex ◽

Hill Coefficient ◽

Phosphate Donor ◽

Km Value ◽

Number Of Binding Sites ◽

The Hill ◽

Kinetics Of

When ATP, the normal phosphate donor for hexokinase D (‘glucokinase’), is replaced by ITP, the positive co-operativity with respect to glucose disappears. This may be rationalized in relation to kinetic models for hexokinase D co-operativity, which assume that with the normal substrates the chemical reaction and subsequent release of products occur so rapidly that binding of substrates cannot approach equilibrium and is therefore not constrained by the thermodynamic requirement that the Hill coefficient for substrate binding cannot exceed the number of binding sites. ITP is a much poorer substrate than ATP, however: its Km value at high glucose concentrations is 24 times the value for ATP, whereas the value of the limiting rate V is decreased about 8-fold. Consequently it is no longer possible for the ternary complex to be converted into products rapidly enough to generate kinetic co-operativity. The negative co-operativity with respect to glucose observed in 2H2O with ATP as phosphate donor also disappears when ITP is used instead of ATP.

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Protamine Neutralization of the Release of Tissue Factor Pathway Inhibitor Activity by Heparins

Thrombosis and Haemostasis ◽

10.1055/s-0038-1649704 ◽

1993 ◽

Vol 70 (06) ◽

pp. 0942-0945 ◽

Cited By ~ 18

Author(s):

Job Harenberg ◽

Marietta Siegele ◽

Carl-Erik Dempfle ◽

Gerd Stehle ◽

Dieter L Heene

Keyword(s):

Tissue Factor ◽

Binding Sites ◽

Tissue Factor Pathway Inhibitor ◽

Factor Xa ◽

Protamine Sulfate ◽

Low Molecular Weight ◽

Factor X ◽

Rebound Phenomenon ◽

Tissue Factor Pathway ◽

Kinetics Of

SummaryThe present study was designed to investigate the action of protamine on the release of tissue factor pathway inhibitor (TFPI) activity by unfractionated (UF) and low molecular weight (LMW) heparin in healthy individuals. 5000 IU UF-heparin or 5000 IU LMW-heparin were given intravenously followed by saline, 5000 U protamine chloride or 5000 U protamine sulfate intravenously after the 10 min blood sample. Then serial blood samples for the measurement of TFPI activity and anti-factor Xa- activity were taken, in order to detect a possible relation between the remaining anti-factor X a activity after neutralization of LMW-heparin with protamine and TFPI activity and to establish whether or not a rebound phenomenon of plasmatic TFPI occurs.There was no difference in the release and in the kinetics of TFPI by UF- and LMW-heparin with subsequent administration of saline. After administration of protamine TFPI activity decreased immediately and irreversibly to pretreatment values. There were no differences between protamine chloride and protamine sulfate on the effect of TFPI induced by UF- or LMW-heparin. No rebound phenomenon of TFPI activity occurred. In contrast anti-factor Xa- activity, as measured by the chromogenic S2222-assay, issued the known differences between UF- and LMW-heparin. The half-life of the aXa-effect of LMW-heparin was twice as long as of UF-heparin. Protamine antagonized UF-heparin completely and about 60% of the anti-factor Xa activity of LMW-heparin, using chromogenic S2222-method. No differences could be detected for protamine chloride and sulfate form of protamineIt is assumed that protamine displaces heparins from the binding sites of TFPI. There were no differences between UF- and LMW-heparin. The data indicate that the sustained antifactor Xa activity after antagonization of LMW-heparins as well as heparin rebound phenomena are not mediated by TFPI activity.

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Abnormal Platelet Serotonin Uptake and Binding Sites in Myeloproliferative Disorders

Thrombosis and Haemostasis ◽

10.1055/s-0038-1661099 ◽

1984 ◽

Vol 51 (03) ◽

pp. 349-353 ◽

Cited By ~ 9

Author(s):

C Caranobe ◽

P Sié ◽

F Fernandez ◽

J Pris ◽

S Moatti ◽

...

Keyword(s):

Binding Sites ◽

High Capacity ◽

Specific Inhibitor ◽

Myeloproliferative Disorders ◽

Normal Subjects ◽

Binding Data ◽

Full Explanation ◽

Number Of Binding Sites ◽

5 Ht Uptake ◽

Kinetics Of

SummaryA simultaneous investigation of the kinetics of serotonin (5 HT) uptake and of binding sites was carried out in the platelets of normal subjects and of 10 patients affected with various types of myeloproliferative disorders (MD). The 5 HT uptake was analysed according to the Lineweaver-Burk and the Eadie-Hofstee methods. With the two methods, the patient’s platelets exhibited a dramatic reduction of the Vi max and of the Km; in some patients the Eadie-Hofstee analysis revealed that a passive diffusion phenomenon is superimposed on the active 5 HT uptake at least for the higher concentration used. The binding data were analysed with the Scatchard method. Two classes of binding sites (high affinity - low capacity, low affinity - high capacity) were found in normal subjects and patients. Pharmacological studies with imipramine, a specific inhibitor of 5 HT uptake, suggested that both the sites are involved in 5 HT uptake. The number of both binding sites was significantly decreased in patient’s platelets while the affinity constants of these binding sites were not significantly reduced in comparison with those of the control subjects. No correlations were found between Vi max, Km and the number of binding sites. These results suggest that a reduction in the number of platelet membrane acceptors for 5 HT commonly occurs in myeloproliferative disorders but does not provide a full explanation of the uptake defect.

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m-[o-(2-Chloro-5-Fluorosulfonylphenylureido)Phenoxybutoxy]Benzamidine: Affinity Labeling Reagent Which Can Identify Secondary Binding Sites of Coagulation Complement and Fibrinolytic Serine Proteases

10.1055/s-0038-1665767 ◽

1979 ◽

Author(s):

D Bing ◽

D Robison ◽

J Andrews ◽

R Laura

Keyword(s):

Binding Sites ◽

Serine Proteases ◽

Enzyme Inhibitor ◽

Molecular Model ◽

Factor Xa ◽

Affinity Labeling ◽

Catalytic Center ◽

Inhibitor Complex ◽

Polypeptide Chains ◽

Kinetics Of

We have determined that m-[o-(2-chloro-5-fluorosulfonylphenylureido)phenoxybutoxy]benza-midine [mCP(PBA)-F] is an affinity labeling reagent which labels both polypeptide chains of thrombin, factor Xa, complement component CIS and plasmin. As this means it is reacting outside of the catalytic center, we have called this reagent an exo-site affinity labeling reagent. Progressive irreversible inhibition of these enzymes by this reagent is rapid (k1st 2.5-4.6 x 10-3sec-1), the kinetics of inactivation are consistent with inhibition proceding via formation of a specific enzyme-inhibitor complex analogous to a Michaelis-Menton complex (KL - 115-26 μM), and diisopropylfluorophosphate or p-amidino-phenylmethanesulfonyfluoride Prevent labeling by [3H]mCP(PBA)-F. A molecular model of mCP(PBA)-F shows that the reactive SO2F group can be 17 A from the cationic amidine. The data are consistent with the hypothesis that both peptide chains are required for the specific proteolytic activity exhibited by these proteases and that the peptide chain which does not contain the active site serine is close to the catalytic center. (Supported by NIH and AHA grants

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