Deviation from Michaelis-Menten kinetics for fumarase

Crabbe, M. James C.

doi:10.1042/bj1570333

Cited by 12 publications

(7 citation statements)

References 6 publications

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“…We re-investigated the conditions described by Crabbe & Bardsley (1976), but could not reproduce their findings. Accordingly we conclude that fumarase is 2:2 or possibly 3:3, and dismiss the earlier claims by Crabbe & Bardsley (1976), which evidently resulted from using an experimental procedure with some unexplained systematic errors.…”

Section: Methodscontrasting

confidence: 64%

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Deviations from Michaelis-Menten kinetics. The possibility of complicated curves for simple kinetic schemes and the computer fitting of experimental data for acetylcholinesterase, acid phosphatase, adenosine deaminase, arylsulphatase, benzylamine oxidase, chymotrypsin, fumarase, galactose dehydrogenase, β-galactosidase, lactate dehydrogenase, peroxidase and xanthine oxidase

Bardsley¹,

Leff²,

Kavanagh³

et al. 1980

Biochemical Journal

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The possible graph shapes for one-site/two-state and substrate-modifier models are discussed. The two-state model is a version of the Monod-Wyman-Changeux model and gives a rate equation with 240 denominator terms. Discussion in terms of K and V effects is not possible. A simplified version of the mechanism can be shown to give v-versus-[S] curves that are either sigmoid or non-sigmoid. They may show substrate inhibition or no final maximum, and the double-reciprocal plots can be concave up or down. The corresponding binding model is determined by only two constants and gives a linear double-reciprocal plot. The substrate-modifier mechanism is a simple example of a mechanism where inclusion of catalytic steps leads to a genuine increase in degree of the rate equation. The v-versus-[S] curve can show such complexities as two maxima and a minimum, and the double-reciprocal plot can cross its asymptote twice, proving the rate equation to be 4:4. A simplified version is 3:3, and analysis shows that at least 18 of the 27 double-reciprocal plots that can arise with 3:3 functions are possible with this particular mechanism. Representative double-reciprocal and Scatchard plots are presented for several sets of rate-constant values. It is concluded that relatively simple mechanisms give pseudo-steady-state rate equations of high degree and considerable complexity. With extended ranges of substrate concentrations there is every reason to believe that experimental data would show the sort of deviations from Michaelis-Menten kinetics seen with calculated curves for such simple mechanisms. Narrow ranges of substrate concentration, on the other hand, would lead to inflexions and curvature being overlooked. It is not possible to discuss such deviations from Michaelis-Menten kinetics in terms of kinetic constants such as Km and V, and, in general, it is also difficult to see any simple way to explain intuitively such features as sigmoidicity, substrate inhibition, double-reciprocal convexity and decrease in degree by cancellation of common factors between numerator and denominator of rate equations. These conclusions apply with even more force when catalytic steps are included, for then the rate equations, are for multi-site mechanisms, of higher degree, allowing increasingly complex curve shapes. A number of enzymes were studied and initial-rate data were fitted by computer.(ABSTRACT TRUNCATED AT 400 WORDS)

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Section: Methodscontrasting

confidence: 64%

“…kinetics (Crabbe & Bardsley, 1976) requiring fourth-degree terms, but this was contested (C. Frieden, personal communication). Accordingly we determined afresh the best-fit curves for fumarase over a wide variety of conditions of pH, temperature, ionic strength and alternative buffer systems.…”

Section: Methodsmentioning

confidence: 99%

Deviations from Michaelis-Menten kinetics. The possibility of complicated curves for simple kinetic schemes and the computer fitting of experimental data for acetylcholinesterase, acid phosphatase, adenosine deaminase, arylsulphatase, benzylamine oxidase, chymotrypsin, fumarase, galactose dehydrogenase, β-galactosidase, lactate dehydrogenase, peroxidase and xanthine oxidase

Bardsley¹,

Leff²,

Kavanagh³

et al. 1980

Biochemical Journal

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“…In fact the observed ratios are approximately 1 : 3 : 9 : 18 : 27 (the last one corresponds to additional binding that inhibits the exchange reaction), i.e. (18) obtained in this way has to be considered with caution and only indicates that the degree is probably higher than 10. The complex with half saturation of catalytic sites seems to have a particular significance as indicated by the rather horizontal plateau that corresponds to it.…”

Section: Model I : a Single Enzymatic Species Catalyses The Exchange mentioning

confidence: 99%

Kinetic and Binding Properties of the Oxoglutarate Translocator of Rat-Heart Mitochondria

et al. 1979

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The kinetic study of the oxoglutarate,,,/malatei, exchange through the inner mitochondrial membrane of rat-heart mitochondria has been completed and extended to higher external-oxoglutarate and to lower internal-malate concentrations. It has been found that the external oxoglutarate inhibits the exchange at high concentration. This excess-substrate inhibition is preceded by four jumps. The kinetic-saturation curve by the internal malate presents an apparent positive cooperativity that may be interpreted in different ways. The independence of the effects of the two substrates on the initial rate has been observed again and supports the conclusions reached in previous work.A method for the determination of oxoglutarate binding to the external face of the inner membrane is described. The binding curve shows four intermediary plateau regions that reflect significant apparent K-effects, alternatively negative and positive.For external-oxoglutarate concentrations below the region of excess-substrate inhibition, the binding-saturation curve and the kinetic-saturation curve are similar, demonstrating that K-effects are predominant. A particularly wide intermediary plateau that seems to correspond to half saturation of the active sites is common to both saturation curves. A clear lack of proportionality between the two curves at low oxoglutarate concentrations seems to indicate that more than one catalytic-rate constant is implied in the exchange kinetics.Two models of the oxoglutarate carrier are presented. Both lead to a minimum degree of 10: 10 for the equation of the binding of oxoglutarate to the catalytic sites. In the first model this corresponds to ten subunits associated into a single oligomer while in the second model this results from a mixture of monomeric, dimeric, trimeric and tetrameric associations.A battery of translocators embedded in the inner mitochondrial membrane catalyses exchange-diffusion of anions between the intramitochondrial and the extramitochondrial compartments following a oneto-one stoichiometry. These translocators show a definite selectivity for the anions exchanged and a specific sensitivity to inhibitors (for a review, see [l]) and couple the metabolic processes occurring inside and outside the mitochondrial matrix.This coupling is subordinated to the properties of the translocators and to the kinetic characteristics of the translocation they take part in; in this way translocators may have a regulatory r81e in cellular metabolism. For example, the oxoglutarate translocator may control the transport of reducing equivalents through the inner mitochondrial membrane of mitochondria via the malate-aspartate shuttle and thus control both internal and external NADH/NAD+ ratios. To conceive valid experiments aimed at testing such a rde, the detailed kinetics and the mode of action of the translocator in situ must be known first.The translocator catalyses various one-to-one exchanges, internal against external substrate ; the concentration of both substrates influences the initial rate in a w...

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“…These findings supported the idea of a two-point attachment of the substrate to the enzyme. More wide-ranging conclusions were drawn from the investigations of Crabbe & Bardsley (1976). Their findings, of kinetics in which the velocity curve descended from the hyperbolic form at substrate concentrations of 6Km and, furthermore, oscillated, lead to the conclusion that a rate equation of at least fourth degree was required for the description and demanded a mixture of isoenzymes for its interpretation.…”

mentioning

confidence: 99%

Lack of deviation from Michaelis–Menten kinetics for pig heart fumarase

Andersen¹

1980

Biochemical Journal

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Studies of steady-state kinetics of fumarase in the usual substrate-concentration range from 0.1 Km to 10 Km and in the high substrate-concentration range from 10 Km to 200 Km are described. The purpose is to investigate reports of substrate inhibition and oscillatory kinetics. In the normal substrate-concentration range, no deviations from hyperbolic kinetics were found, and in the extended concentration range, up to more than 200 times the Km value, no substrate inhibition was demonstrated. A discussion of the discrepancies between the mentioned reports of deviations from the hyperbolic kinetics and the present findings is given.

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Deviation from Michaelis-Menten kinetics for fumarase

Cited by 12 publications

References 6 publications

Kinetic and Binding Properties of the Oxoglutarate Translocator of Rat-Heart Mitochondria

Lack of deviation from Michaelis–Menten kinetics for pig heart fumarase

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