A kinetic study of baker's-yeast pyruvate kinase activated by fructose 1,6-diphosphate

MacFarlane, Neil; Ainsworth, Stanley

doi:10.1042/bj1291035

Cited by 49 publications

(50 citation statements)

References 23 publications

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“…The concentrations ofthe different species involved have been determined from the known equilibrium constants for their inter-conversions by using the method described by Macfarlane & Ainsworth (1972). This examination showed that five ADP and four phosphoenolpyruvate species are present in significant proportions at pH6.2: their identity and contributions to the sum of the free and The total Mg2" concentration contains an addition for Mg2+-bound ATP when this product is present as an inhibitor.…”

Section: Theory Substrate-level Equilibriamentioning

confidence: 99%

“…We propose to determine whether the free or Mg2+-complexed forms of ADP and phosphoenolpyruvate are the true substrates of muscle pyruvate kinase, by using the test devised by Macfarlane & Ainsworth (1972). The basis of the test is to measure the reciprocal velocity as a function of Mg2+ concentration under conditions where the concentrations of different combinations of possible substrates are kept constant, and to estimate thereby the powers of Mg2+ concentration which appear in the empirical quaternary complex of the substrates, with the random dissociation of pyruvate and MgATP.…”

Section: Identification Ofsubstratesmentioning

confidence: 99%

“…Cleland (1967a) has questioned whether the bivalent metal cation and ADP can bind separately to the enzyme and has stated that nucleoside di-and tri-phosphates always react in the form of their bivalent metal-ion complexes. This does not seem certain, however, for Macfarlane & Ainsworth (1972) have shown that free ADP alone is the nucleotide substrate of yeast pyruvate kinase.…”

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confidence: 99%

“…The reactions took place at pH6.2 and 25°C; K+ was present at all times at a saturating concentration (Macfarlane & Ainsworth, 1972).…”

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A kinetic study of rabbit muscle pyruvate kinase

Ainsworth

MacFarlane

1973

Biochemical Journal

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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.

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Section: Theory Substrate-level Equilibriamentioning

confidence: 99%

Section: Identification Ofsubstratesmentioning

confidence: 99%

mentioning

confidence: 99%

“…The reactions took place at pH6.2 and 25°C; K+ was present at all times at a saturating concentration (Macfarlane & Ainsworth, 1972).…”

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confidence: 99%

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A kinetic study of rabbit muscle pyruvate kinase

Ainsworth

MacFarlane

1973

Biochemical Journal

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“…The enzymes, which have been isolated from mammalian tissues (Tietz & Ochoa, 1958;Irving & Williams, 1973;Nicholas & Bachelard, 1974) (Reynard et al, 1961;Ainsworth & MacFarlane, 1973), an ordered addition of substrates and dissociation of products, for the yeast enzyme (MacFarlane & Ainsworth, 1972) and a Ping-Pong mechanism involving a phosphoenzyme for the liver L-type enzyme (MacFarlane & Ainsworth, 1974). There are difficulties, however, in the interpretation of initial-velocity studies, and we are therefore studying the mechanism of these enzymes with a variety of techniques, including flux measurements with labelled substrates (Britton, 1966;, isotope-trapping (Rose et al, 1974), binding and initial-velocity studies.…”

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Kinetics and mechanism of action of muscle pyruvate kinase

Dann

Britton

1978

Biochemical Journal

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1. The mechanism of rabbit muscle pyruvate kinase was investigated by measurements of fluxes, isotope trapping, steady-state velocity and binding of the substrates. All measurements were made at pH8.5 in Tris/HCl buffer and at 5mM-free Mg2+. ADP/ Flux of ATP to phosphoenolpyruvate (measured at equilibrium) increased hyperbolically with ADP concentration from unity to about 2.1 at 2mm-ADP, but was unaffected by phosphoenolpyruvate concentration. Since the ratio is greater than unity, one pathway for the addition of substrates must involve phosphoenolpyruvate adding first to the enzyme in a rate-limiting step. However, the substrates must also add in the alternative order, because of the non-linear increase in the ratio with ADP concentration and because the rate of increase is very much less than that predicted from the steady-state velocity data for an ordered addition. The lack of influence of phosphoenolpyruvate on the ratio is consistent with the rapid addition of ADP in the alternative pathway. At low ADP concentrations the alternative pathway contributes less than 33 % to the total reaction. 4. Isotope trapping was observed with [32P]phosphoenolpyruvate, confirming that when phosphoenolpyruvate adds first to the enzyme it is in a rate-limiting step. The release of phosphoenolpyruvate from the ternary complex must also be a slow step. Trapping was not observed with [8-14C]ADP, hence the addition of ADP to the free enzyme must be rapid unless its dissociation constant is very large (>20mM). 5. Binding studies showed that 4mol of [32P]phosphoenolpyruvate binds to 1 mol ofthe enzyme, probably unligated to Mg2+, with a dissociation constant appropriate to the mechanism indicated above. Binding of [8-14C]ADP could not be detected, and hence the binding of ADP occurs by a low-affinity step. The latter is also demanded by the steady-state velocity data. 6. The ratio Flux of phosphoenolpyruvate to ATP/Flux of phosphoenolpyruvate to pyruvate (determined from the incorporation of label into phosphoenolpyruvate from [3-14C]-pyruvate or [y-32P]ATP during the forward reaction) did not differ significantly from unity. Steady-state velocity data predicted grossly different flux ratios for ordered dissociations of the products, and the results indicate that the dissociation must be rapid and random. The data also exclude a Ping-Pong mechanism. 7. Permissible rate constants for the above mechanism are calculated. The results indicate a high degree of cooperativity in binding, whatever the order of addition of substrate.Pyruvate kinase (EC 2.7.1.40) catalyses the formation of pyruvate and ATP from phosphoenolpyruvate and ADP and is an important enzyme in the regulation ofglycolysis and gluconeogenesis (Llorente et al.,

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Solute compatibility with enzyme function and structure: Rationales for the selection of osmotic agents and end‐products of anaerobic metabolism in marine invertebrates

1979

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The major nitrogenous osmolytes present in the cells of marine invertebrates, notably the free amino acids glycine, alanine and proline, and trimethylamine oxide and betaine, are highly compatible with proper enzyme function and structure. These nitrogenous osmolytes display either non-perturbing or, in some cases, favorable effects on enzyme-substrate and enzyme-cofactor complex formation, catalytic velocity and protein structural stability. In contrast, inorganic salts (KCl and NaCl) and certain of the free amino acids which play only a minor osmotic role, e.g., arginine and lysine, have strongly perturbing effects on one or more of these enzymic parameters. The compatible nitrogenous solutes therefore are suitable for use at high (several tenths molar) concentrations and at widely varying concentrations in osmo-conforming species. Certain nitrogenous solutes, especially trimethylamine oxide, betaine and glutamate, offset some of the perturbing effects of inorganic ions on enzyme function. The selective accumulation of osmolytes thus involves not only the concentration of non-perturbing solutes, but also a balanced accumulation of solutes with opposing effects on enzymes. The selection of end-products of anaerobic metabolism also appears to be based, in part, on considerations of solute compatibility with enzyme function. Octopine is a non-perturbing solute, whereas arginine, which is condensed with pyruvate to form octopine, is very strongly perturbing. Succinate has marked stabilizing effects on protein structure. We conclude that the composition of the intracellular fluids of marine invertebrates reflects selection for osmolytes and end-products whose net effects create a cellular microenvironment which is conducive to optimal enzyme function and structure. The accumulation of compatible solutes may preclude the necessity for widespread changes in protein structure in adapting to concentrated or highly variable osmotic environments.

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A kinetic study of baker's-yeast pyruvate kinase activated by fructose 1,6-diphosphate

Cited by 49 publications

References 23 publications

A kinetic study of rabbit muscle pyruvate kinase

A kinetic study of rabbit muscle pyruvate kinase

Kinetics and mechanism of action of muscle pyruvate kinase

Solute compatibility with enzyme function and structure: Rationales for the selection of osmotic agents and end‐products of anaerobic metabolism in marine invertebrates

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