The interpretation of kinetic data for enzyme-catalysed reactions involving three substrates

Dalziel, Keith

doi:10.1042/bj1140547

Cited by 141 publications

(83 citation statements)

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“…[acceptor], and consequently the values for 41 obtained by the present method would be the true values [16]. Such a mechanism was proposed by Orsi and Cleland [7] for rabbit muscle glyceraldehyde-3-phosphate dehydrogenase.…”

Section: Discussionsupporting

confidence: 64%

“…In principle, alternative mechanisms for threesubstrate reactions can be distinguished by steadystate measurements with widely varied concentrations of all three substrates [16]. When there is substrate inhibition, however, and when slope changes over accessible ranges of substrate concentration are small as in the present case, the discrimination of the method is low, as was discussed by Keleti et al [17].…”

Section: Discussionmentioning

confidence: 80%

“…Consideration of the rate equations for threesubstrate reaction mechanisms [16] shows that the values for 4", 41 and 4 2 obtained in this way will, in general, be larger than the true values, although with arsenate in particular the difference will be small because a concentration o f 5 mM is close to saturating (Fig1 A). However, in the case of an ordered mechanism in which NAD' is the first and acceptor the last substrate to combine, the initial rate equation lacks the term ~I~/ [ N A D ' ]…”

Section: Discussionmentioning

confidence: 90%

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Kinetic Studies of Glyceraldehyde‐3‐Phosphate Dehydrogenase from Rabbit Muscle

Meunier¹,

Dalziel²

1978

European Journal of Biochemistry

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Initial rate studies at pH 7.6 with three aldehydes, product inhibition patterns with NADH and dead-end inhibition with adenosine diphosphoribose show that the kinetic mechanism of glyceraldehyde-3-phosphate dehydrogenase from rabbit muscle cannot be ordered, and support an enzyme-substitution mechanism. Deviations from Michaelis-Menten behaviour are consistent with negative interactions in the binding of NAD' and instability of the species E(NAD)3 and E(NAD)4. Inhibition with large concentrations of phosphate and arsenate indicates competition for a binding site for glyceraldehyde 3-phosphate, and is not found with glyceraldehyde as substrate.I t is known that the binding of NAD' or NADH to rabbit muscle glyceraldehyde-3-phosphate dehydrogenase (~-glyceraldehyde-3-phosphate : NAD + oxidoreductase, phosphorylating) involves more than one dissociation constant, indicating either that the four subunits of the enzyme are not identical and equivalent, or that the coenzyme binding is negatively cooperative [I -31. The greatest difference of binding free energy is between the second and third coenzyme molecules [3] and there is evidence of a conformational change of the protein at half-saturation with NAD' [4].The steady-state kinetics of the enzyme-catalysed reaction have been investigated in some detail by several groups of workers, with conflicting results and conclusions. Furfine and Velick [5] concluded that mechanism was of the rapid equilibrium, random type involving a quaternary complex, from data with glyceraldehyde 3-phosphate as substrate, whereas Fahien [6], using glyceraldehyde, proposed an ordered mechanism. Orsi and Cleland [7] also proposed an ordered mechanism with NAD', aldehyde and phosphate or arsenate adding in that order, based on studies of inhibition by substrate, products and inactive substrate analogues, and suggested that release of NADH is the final step, rate-limiting when glyceraldehyde 3-phosphate is the substrate. In none of ~ -Ahh,rviarion.s. Gra-3-P, glyceraldehyde 3-phosphate; Gra, Etiqwe. Glyceraldehyde-3-phosphate dehydrogenase (EC glyceraldeh yde. 1.2.1.12).these studies, nor in similar studies with the pigmuscle enzyme [8,9], were deviations from MichaelisMenten behaviour suggestive of negative cooperativity reported. Intersecting patterns of linear reciprocal plots were obtained, which also appeared to eliminate the possibility of an enzyme-substitution mechanism with product dissociation before combination of all the substrates.However, Duggleby and Dennis [lo] have since reported initial rate data for the enzyme from pea seeds consistent with an enzyme-substitution mechanism in which the two stable forms of enzyme involved in the catalytic cycle are an enzyme . NAD' complex and acyl-enzyme. They argued that this is the basic mechanism for the enzyme from other sources also. A similar mechanism had been proposed by Trentham [ll] from pre-steady-state kinetic studies of the enzymes from lobster and sturgeon muscle.The objectives of the present work were to reinvestigate the st...

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Section: Discussionsupporting

confidence: 64%

Section: Discussionmentioning

confidence: 80%

Section: Discussionmentioning

confidence: 90%

See 1 more Smart Citation

Kinetic Studies of Glyceraldehyde‐3‐Phosphate Dehydrogenase from Rabbit Muscle

Meunier¹,

Dalziel²

1978

European Journal of Biochemistry

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“…A range of six suitable concentrations of each substrate was chosen. Results were analyzed graphically as described by Dalziel [27] using linear regression analysis. The substrate concentrations were determined by enzymatic spectrophotometric methods.…”

Section: Assays For Malate Dehydrogenasementioning

confidence: 99%

“…Data were analyzed and described by Dalziel [27]. Substrate coefficients were estimated at the 99% level of significance The effect of pH variation on malate dehydrogenase activity in both directions was studied.…”

Section: K Values Of Malate Dehydrogenasefractionsmentioning

confidence: 99%

Identification of cytoplasmic nodule‐associated forms of malate dehydrogenase involved in the symbiosis between Rhizobium leguminosarum and Pisum sativum

Appels¹,

Haaker²

1988

European Journal of Biochemistry

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The malate dehydrogenase activity (EC 1.1.1.37), present in the cytoplasm of Pisum sativum root nodules, can be separated by ion-exchange chromatography into four different fractions. Malate dehydrogenase activity present in the cytoplasm of roots elutes mainly as a single peak.During nodule development an increase in malate dehydrogenase activity per gram of material was observed. This increase occurred concomitantly with the increase in nitrogenase activity.The kinetic properties of the separated malate dehydrogenases of root nodule cytoplasm and root cytoplasm were studied. The K , values for malate (2.6 mM), NAD' (27 pM), oxaloacetate (18 pM) and NADH (13 pM) of the dominant form of the root nodule cytoplasm are much lower than those of the dominant malate dehydrogenase root form (64 mM, 4.4 mM, 89 pM and 70 pM respectively). Binding of malate by the enzyme-NADH complex from noot nodules results in an abortive complex, thereby blocking the further reduction of oxaloacetate by NADH. The dominant root malate dehydrogenase does not form the abortive complex.From the kinetic data it is concluded, first, that the root nodule forms of the enzyme are capable of catalysing at a high rate the reduction of oxaloacetate, to meet the demands for malate governed by the bacterioid and the infected plant cell. The second conclusion, drawn from the kinetic data, is that under physiological conditions the conversion of oxaloacetate can be controlled just by the malate concentration.Consequently the major root nodule forms of malate dehydrogenase are able to allow a high flux of malate production from oxaloacetate but also to establish a sufficient oxaloacetate concentration necessary for the assimilation and transport of fixed nitrogen.In the absence of an adequate supply of available soil nitrogen, certain leguminous species are capable of forming symbiotic associations with soil bacteria of the genus Rhizobium. The intracellular symbiotic association arises in the cortex of legume roots following infection by Rhizobium. In the root cortex the bacteria cause formation of meristems. The bacteria penetrate the newly divided host cells through growth and branching of the infection threads. Released bacteria are enclosed by envelopes of host cell plasmalemma and develop into bacteroids. This differentiated form of bacteria is capable of the reduction of N2 to NH3 [I]. The continued division of infected cells and the proliferation of bacterial cells result in the formation of root nodules with a specific morphological arrangement (for a review see [2]).To allow nitrogen fixation to occur in the root nodule there must be an extensive metabolic balance between bacteroids and the different plant cells. Photosynthetic products of the host are used by the plant cells and bacteroids. Dioxygen, transported by leghemoglobin, together with carbon compounds made available to the bacteroids, are used to generate reducing power and ATP for nitrogen fixation. The product of the nitrogenase reaction, NH3, is assimilated in the cytosol of the ...

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Kinetics of Catalyzed Reactions – Biological

McDonald

Tipton

2002

Encyclopedia of Catalysis

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An understanding of the kinetic reaction pathways (mechanisms) followed by enzymes is an essential prerequisite to understanding how enzymes might function in their normal complex metabolic enviroments within cells and tissues. It is also important for the interpretation of the behavior of mutant enzymes, the design and behavior of enzyme inhibitors for drug use, and the meaningful comparison of enzyme activities between laboratories, tissue, and individuals. This account describes the kinetic behavior of enzymes that catalyze reactions involving one or more substrates, the mechanisms that may be followed and the application of inhibition and other approaches to determining the mechanism followed. Systems obeying simple saturation kinetics are described together with those that may lead to more complex behavior. Enzyme classification and nomenclature are outlined and possible complexities in the initial‐rate behavior of enzyme‐catalyzed reactions and in the dependence of rate upon enzyme concentration are also described along with accepted standards for the expression of enzyme activities.

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The interpretation of kinetic data for enzyme-catalysed reactions involving three substrates

Cited by 141 publications

References 10 publications

Kinetic Studies of Glyceraldehyde‐3‐Phosphate Dehydrogenase from Rabbit Muscle

Kinetic Studies of Glyceraldehyde‐3‐Phosphate Dehydrogenase from Rabbit Muscle

Identification of cytoplasmic nodule‐associated forms of malate dehydrogenase involved in the symbiosis between Rhizobium leguminosarum and Pisum sativum

Kinetics of Catalyzed Reactions – Biological

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