scholarly journals Selective modification of rabbit muscle pyruvate kinase by 5-chloro-4-oxopentanoic acid

1976 ◽  
Vol 159 (2) ◽  
pp. 201-211 ◽  
Author(s):  
D P Bloxham ◽  
R A Chalkley
Keyword(s):  
Pyruvate Kinase ◽  
Active Site ◽  
Functional Group ◽  
Time Dependent ◽  
Rabbit Muscle ◽  
Pentanoic Acid ◽  
Basic Group ◽  
Muscle Pyruvate Kinase ◽  
Kinetics Of ◽  
Kinetics Of Inhibition

Rabbit muscle pyruvate kinase was irreverisbly inactivated by 5-chloro-4-oxopentanoic acid with a pKa of 9.2. The inhibition was time-dependent and was related to the 5-chloro-4-oxopentanoic acid concentration. Analysis of the kinetics of inhibition showed that the binding of the inhibitor showed positive co-operativity (n = 1.5 ± 0.2). Inhibition of pyruvate kinase by 5-chloro-4-oxopentanoic acid was prevented by ligands which bind to the active site. Their effectiveness was placed in the order Mg2+ > phosphoenolpyruvate > ATP ≫ ADP > pyruvate. Inhibitor-modified pyruvate kinase was unable to catalyse the detritiation of [3-3H]pyruvate in the ATP-promoted reaction, but it did retain 5-10% of the activity with either phosphate or arsenate as promoters. 5-Chlor-4-oxo-[3,5-3H]pentanoic acid was covalently bound to pyruvate kinase and demonstrated a stoicheiometry of 1 mol of inhibitor bound per mol of pyruvate kinase protomer. The incorporation of the inhibitor and the loss of enzyme was proportional. These results are discussed in terms of 5-chloro-4-oxopentanoic acid alkylating a functional group in the phosphoryl overlap region of the active site, and a model is presented in which this compound alkylates an active-site thiol in a reaction that is controlled by a more basic group at the active site.

scholarly journals A kinetic study of rabbit muscle pyruvate kinase

1973 ◽  
Vol 131 (2) ◽  
pp. 223-236 ◽  
Author(s):  
S. Ainsworth ◽  
N. Macfarlane
Keyword(s):  
Reaction Mechanism ◽  
Kinetic Study ◽  
Pyruvate Kinase ◽  
Random Order ◽  
Experimental Results ◽  
Rabbit Muscle ◽  
Dead End ◽  
Inhibition Constants ◽  
Muscle Pyruvate Kinase ◽  
Kinetics Of

The paper reports a study of the kinetics of the reaction between phosphoenolpyruvate, ADP and Mg2+ catalysed by rabbit muscle pyruvate kinase. The experimental results indicate that the reaction mechanism is equilibrium random-order in type, that the substrates and products are phosphoenolpyruvate, ADP, Mg2+, pyruvate and MgATP, and that dead-end complexes, between pyruvate, ADP and Mg2+, form randomly and exist in equilibrium with themselves and other substrate complexes. Values were determined for the Michaelis, dissociation and inhibition constants of the reaction and are compared with values ascertained by previous workers.

scholarly journals Identification of cysteine as the reactive group in pyruvate kinase alkylated by 5-chloro-4-oxopentanoic acid

1976 ◽  
Vol 159 (2) ◽  
pp. 213-219 ◽  
Author(s):  
R A Chalkley ◽  
D P Bloxham
Keyword(s):  
Pyruvate Kinase ◽  
Peptide Mapping ◽  
Periodate Oxidation ◽  
Performic Acid ◽  
Acid Oxidation ◽  
Rabbit Muscle ◽  
Pentanoic Acid ◽  
Reactive Group ◽  
Muscle Pyruvate Kinase ◽  
Nabh4 Reduction

4-Hydroxypentanoic acid alanine thioether was synthesized and characterized by n.m.r. spectroscopy. This derivative corresponded to the modified amino acid obtained by allowing 5-chloro-4-oxo[3,5-3H]pentanoic acid to react with rabbit muscle pyruvate kinase. Performic acid oxidation of 4-oxo[3,5-3H]pentanoic acid alanine thioether in pyruvate kinase gave [3H]succinate (67%) and [3H]carboxymethylcysteine (33%) as expected. Evidence is presented to show that NaBH4 reduction followed by periodate oxidation and analysis of radioactive formaldehyde production may provide a convenient method for distinguishing between thiol and amino alkylation by halogenomethyl ketone compounds. Peptide ‘mapping’ confirms that the modification by 5-chloro-4-oxopentanoic acid occurs primarily at one region of pyruvate kinase.

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

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.

Isolation and sequence determination of an active site peptide of rabbit muscle pyruvate kinase

1987 ◽  
Vol 253 (1) ◽  
pp. 133-137 ◽  
Author(s):  
Guillermo Bezares ◽  
Jaime Eyzaguirre ◽  
Maria Victoria Hinrichs ◽  
Robert L. Heinrikson ◽  
Ilene Reardon ◽  
...  
Keyword(s):  
Pyruvate Kinase ◽  
Active Site ◽  
Rabbit Muscle ◽  
Sequence Determination ◽  
Muscle Pyruvate Kinase
Sign in / Sign up

Export Citation Format

Share Document