scholarly journals The regulatory properties of yeast pyruvate kinase. Effects of NH4+ and K+ concentrations

1986 ◽  
Vol 234 (3) ◽  
pp. 705-715 ◽  
Author(s):  
N Rhodes ◽  
C N Morris ◽  
S Ainsworth ◽  
J Kinderlerer
Keyword(s):  
Pyruvate Kinase ◽  
Dissociation Constants ◽  
Exponential Model ◽  
Positive Interactions ◽  
Data Sets ◽  
Positive Interaction ◽  
Simultaneous Activation ◽  
Regulatory Properties ◽  
Kinetics Of

The kinetics of pyruvate kinase from Saccharomyces cerevisiae were studied at 25 degrees C and pH 6.2 as a function of the concentrations of ADP, phosphoenolpyruvate, Mg2+ and either NH4+ or K+. The data were analysed by the exponential model for four substrates, obtained by extension of the model described by Ainsworth, Kinderlerer & Gregory [(1983) Biochem. J. 209, 401-411]. On that basis, it was concluded that NH4+ binding is almost non-interactive but leads to the appearance of positive interaction in the velocity response to increase in its concentration because of positive interactions with phosphoenolpyruvate and Mg2+. The data obtained with K+ lead to the same conclusions and differ only in suggesting that NH4+ is bound more strongly to the enzyme than is K+. Both data sets are used as the basis for a discussion of the substrate interactions of pyruvate kinase and it appears therefrom that the heterotropic interactions accord with what is known of the events that take place at the active site during catalysis. The paper also reports a determination of the dissociation constants for the NH4+ complexes with ADP and phosphoenolpyruvate and an examination of the simultaneous activation of pyruvate kinase by K+ and NH4+ ions.

scholarly journals The regulatory properties of yeast pyruvate kinase

1984 ◽  
Vol 217 (3) ◽  
pp. 641-647 ◽  
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.

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

1983 ◽  
Vol 209 (2) ◽  
pp. 401-411 ◽  
Author(s):  
S Ainsworth ◽  
J Kinderlerer ◽  
R B Gregory
Keyword(s):  
Pyruvate Kinase ◽  
Exponential Model ◽  
Rabbit Muscle ◽  
Catalysed Reaction ◽  
Dead End ◽  
Influence Of Substrate ◽  
Muscle Pyruvate Kinase ◽  
Regulatory Properties ◽  
Kinetics 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.

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

1986 ◽  
Vol 234 (3) ◽  
pp. 691-698 ◽  
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.

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

1986 ◽  
Vol 234 (3) ◽  
pp. 699-703 ◽  
Author(s):  
J Kinderlerer ◽  
S Ainsworth ◽  
C N Morris ◽  
N Rhodes
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Pyruvate Kinase ◽  
Exponential Model ◽  
Effect Of Ph ◽  
Ph Range ◽  
Regulatory Properties ◽  
Kinetics Of

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+.

Leishmania pyruvate kinase: the crystal structure reveals the structural basis of its unique regulatory properties

2000 ◽  
Vol 28 (2) ◽  
pp. 186-190 ◽  
Author(s):  
L. A. Fothergill-Gilmore ◽  
D. J. Rigden ◽  
P. A. M. Michels ◽  
S. E. V. Phillips
Keyword(s):  
Crystal Structure ◽  
Pyruvate Kinase ◽  
Regulatory Role ◽  
Structural Basis ◽  
Protozoan Parasites ◽  
Effector Molecule ◽  
T State ◽  
Regulatory Properties ◽  
Allosteric Activator

Glycolysis occupies a central role in cellular metabolism, and is of particular importance for the catabolic production of ATP in protozoan parasites such as Leishmania and Trypanosoma. In these organisms pyruvate kinase plays a key regulatory role, and is unique in responding to fructose 2,6-bisphosphate as allosteric activator. The determination of the crystal structure of the first eukaryotic pyruvate kinase in the T-state (the inactive or ‘tense’ conformation of allosteric enzymes) is described. A comparison of the effector sites of the Leishmania and yeast enzymes reveals the structural basis for the different effector specificity. Two loops, comprising residues 443–453 and 480–489, adopt very different conformations in the two enzymes, and Lys-453 and His-480 that are a feature of trypanosomatid enzymes provide probable ligands for the 2-phospho group of the effector molecule. These and other differences offer an opportunity for the design of drugs that would exploit regulatory differences between parasite and host.

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.

scholarly journals The dependence of DNase I activity on the conformation of oligodeoxynucleotides

1997 ◽  
Vol 321 (2) ◽  
pp. 481-486 ◽  
Author(s):  
Deborah H. SUTTON ◽  
Graeme L. CONN ◽  
Tom BROWN ◽  
Andrew N. LANE
Keyword(s):  
Dissociation Constants ◽  
Minor Groove ◽  
Dnase I ◽  
Optical Signals ◽  
Short Oligonucleotide ◽  
A Value ◽  
Dna Strands ◽  
Kinetics Of ◽  
Continuous Assay

We have developed a sensitive continuous assay for nucleases using proton release. The assay has been applied to the determination of the kinetics of DNase I acting on short, defined deoxyoligonucleotides. The dependence of kcat/Km on sequence and structure of short oligonucleotide substrates has been measured: increasing lengths of AnTn sequences decrease the rate of cleavage. GƃA mismatches in which the bases pair using imino protons are cleaved quite effectively by DNase I. In contrast, tandem GƃA mismatches which use amino pairing and have BII phosphodiesters, are refractory to DNase I. Also, the DNA strands of DNAƃRNA hybrid duplexes are not cleaved by DNase I. These results show that the global conformation of a duplex and the details of its minor groove affect the cleavage efficiency by DNase I. The assay has also been used to measure the inhibition constant of the minor-groove-binding ligand propamidine. A value of 3 ƁM has been determined for binding to the sequence d(CGCGAATTCGCG)2, showing that dissociation constants can be determined even when there are no convenient optical signals for titrations.

Modification of the regulatory properties of pyruvate kinase of Neurospora by growth at elevated temperatures

1976 ◽  
Vol 54 (5) ◽  
pp. 398-407 ◽  
Author(s):  
M. Kapoor ◽  
M. O'Brien ◽  
A. Braun
Keyword(s):  
Complex Formation ◽  
Pyruvate Kinase ◽  
Isoelectric Point ◽  
Elevated Temperatures ◽  
Dissociation Constants ◽  
Allosteric Site ◽  
Allosteric Effector ◽  
Low Concentrations ◽  
Weak Binding ◽  
Regulatory Properties

Pyruvate kinase (EC 2.7.1.40) was isolated from Neurospora crassa mycelium grown at 28 °C (PK-28) and at 42 °C (PK-42). The regulatory properties, particularly the response towards the allosteric effector fructose 1,6-diphosphate (FDP), was different in the two enzymes. PK-28 showed an activation by FDP but PK-42, under comparable conditions, appeared to be activated by low concentrations of FDP and inhibited by higher ones. For PK-28, complex formation with FDP results in a lowering of the isoelectric point from 6.40 to 5.50, representing the pI of the unliganded enzyme and that of the complex, respectively. In contrast to this, PK-42 exhibits a weak binding to FDP as suggested by a lack of decrease in the isoelectric point on treatment with comparable concentrations of FDP. Studies with quenching of aromatic residue fluorescence of PK-28 and PK-42, following binding of FDP, indicate that although this ligand binds to both types of enzymes the affinity for the two is vastly different. Dissociation constants of 9.3 μM and 0.1 mM were calculated for the binding of FDP to PK-28 and PK-42, respectively. It is concluded that growth at elevated temperatures induced a conformational change in the pyruvate kinase leading to partial desensitization of the allosteric site. The nature of the factor(s) responsible for this change is not understood at present.

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