Studies on Liver Alcohol Dehydrogenase. II. The Kinetics of the Compound of Horse Liver Alcohol Dehydrogenase and Reduced Diphosphopyridine Nucleotide.

Theorell, Hugo; Chance, Britton; Holtermann, Hugo; Sörensen, Jörgine Stene; Sørensen, Nils Andreas

doi:10.3891/acta.chem.scand.05-1127

Cited by 448 publications

(114 citation statements)

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“…This mechanism was first proposed for liver alcohol dehydrogenase by Theorell & Chance (1951), and the steady-state rate equation for this mechanism can be written in the form of eqn. (8) (Cleland, 1963b) (8) where K. and Kq are Michaelis constants for products P and Q respectively and K,P and Kjq are inhibition constants for products P and Q respectively.…”

Section: Discussionmentioning

confidence: 99%

Kinetic studies of formate dehydrogenase

Peacock

Boulter

1970

Biochemical Journal

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1. The kinetic mechanism of formate dehydrogenase is a sequential pathway. 2. The binding of the substrates proceeds in an obligatory order, NAD+ binding first, followed by formate. 3. It seems most likely that the interconversion of the central ternary complex is extremely rapid, and that the rate-limiting step is the formation or possible isomerization of the enzyme-coenzyme complexes. 4. The secondary plots of the inhibitions with HC03-and NO3-are non-linear, which suggests that more than one molecule of each species is able to bind to the same enzyme form. 5. The rate of the reverse reaction with carbon dioxide at pH 6.0 is 20 times that with bicarbonate at pH 8.0, although no product inhibition could be detected with carbon dioxide. The low rate of the reverse reaction precluded any steady-state analysis as the enzyme concentrations needed to obtain a measurable rate are of the same order as the Km values for NAD+ and NADH.Formate dehydrogenase (formate-NAD+ oxidoreductase; EC 1.2.1.2) catalyses the reaction: NAD++HC02-÷ NADH+C02and is known to occur in animals, higher plants and bacteria. The higher-plant enzyme differs from both the animal enzyme, which is activated by ATP (Mathews & Vennesland, 1950), and the bacterial enzyme, which does not require NAD+ (Gale, 1939). Although Davison (1951) and Nason & Little (1955) have both reported the effects of various enzyme inhibitors on the activity of the plant enzyme, no kinetic investigation has so far been undertaken.The present study is an investigation into the mechanism of enzyme action by steady-state kinetics. Various enzyme-substrate/enzyme-inhibitor complexes and their possible pathways of interconversion were determined by using initialvelocity, product-inhibition and dead-end-inhibition analysis. EXPERIMENTALEnzyme. Formate dehydrogenase was purified from Phaseolu8 aureu? (mung bean) by a combination of salt fractionation, ion-exchange, hydroxyapatite and aluminagel chromatography, and assayed as described by Peacock (1970). The molecular weight was estimated by gel filtration as 92000I10000, and the enzyme was not more than 50% pure, as determined by acrylamide-gel electrophoresis. Sub8trates. Analytical-grade ammonium formate and NaHCO3 were from British Drug Houses Ltd., Poole, Dorset, U.K.Initial-rate measurements. The reaction was followed by measuring the appearance of NADH at 340nm by using a Unicam SP. 800 spectrophotometer with an attached lOmV 'slave' recorder. The full-scale deflexion of the recorder was set to correspond to 0.1 extinction unit, and the chart speed adjusted to give recorded traces with an angle of inclination of between 30 and 600. Silica cells (4cm light-path) were filled with all the components of the reaction mixture except the enzyme, in a total volume of 9.9ml, and preincubated at 250C. The reaction was initiated by the addition of 0.1ml of suitably diluted enzyme and the cell was immediately placed in the reaction compartment (kept at 25°C). The recorded traces obtained were extrapolated to the time of addition, a...

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Section: Discussionmentioning

confidence: 99%

Kinetic studies of formate dehydrogenase

Peacock

Boulter

1970

Biochemical Journal

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“…so that release of the first product is concerted with binding of the second substrate. This type of mechanism can easily be envisaged for an enzyme such as alcohol dehydrogenase that catalyses a simple reaction at a unique site, the case originally considered by Theorell and Chance (1951), but it is less easily reconciled with a complex enzyme like nitrate reductase that involves communication between chemical events at several Fe-S centres and a molybdenum cofactor. Moreover, the fact that the .…”

Section: Phranlerelmodelmentioning

confidence: 99%

Kinetic Studies of a Soluble αβ Complex of Nitrate Reductase A from Escherichia Coli

Buc

Santini

Blasco

et al. 1995

European Journal of Biochemistry

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A soluble up complex of nitrate reductase can be obtained from a strain of Escheri(:h:hia r:oli that lacks the narl gene and expresses only the n and p subunits. The subunit contains four Fe-S centres and the a subunit contains the molybdenum cofactor, which is the site at which nitrate is reduced. Dcspite the lack of the y subunit of the complete enzyme, this complex can still catalyse the reduction of niirate with artificial electron donors such as benzyl viologen, so that it is suitable for studying the transfer of electrons between these two types of redox centre.To examine whether the electrons from reduced benzyl viologen are initially delivered to the Fe-S centres, or directly to the molybdenuin cofactor, or both, we have studied the stcady-state kinetics and the binding of benzyl viologen to the ap complex and mutants @* with altered 11 subunits.Reduction of the enzyme by reduced benzyl viologen in the absence of nitrate showed that all four Fe-S centres and the molybdenum cofactor could be reduced. Two classes of site with different equilibrium constants could be distinguished.The kinetic results suggest that benzyl viologen supplics its electrons directly to the molybdenum cofactor, at a rate showing a hyperbolic dependence on ihe square of the concentration of the electron donor. A reaction mechanism is proposed for the reduction of nitrate catalysed by the (ID complex of nitrate reductase with artificial electron donors.Keyword..r: nitrate reductase; iron-sulfur centers; molybdenum cofactor ; kinetics.Nitrate can serve as the terminal electron acceptor during anaerobic growth of Escherichia cnli, and nitrate reductase, the enzyme that catalyses the reduction of nitrate, is a memhranebound molybdoenzyme with three dificrent subunits designated IK, fi and 11, coded by the genes narc, niirH and narl, respectivcly. Each of these subunits carries a different redox centre (Rlasco et al., 1989; Guigliarelli et al., 1992): the Q subunit carries a cofactor containing molybdenum at the active centre for the reduction of nitrate to nitrite; the p subunit contains four Fe-S centres and the y subunit is a h-type cytochrome. The (I and subunits form an r $ complex that is bound to the membrane by its interaction with the y subunit embedded in the membrane. The physiological electron source for nitrate reduction is the quinone pool; electrons are transferred initially to the h-type haem, and the complete sequence is postulated to be as follows:Detailed EPR studies have unambiguously indicated the presence of one 13Fe-4SJ and threc 14Fe-4SI centres in the enzyme. These have been named as centres 1, 2, 3 and 4, with redox potentials t X O mV, +60 mV, -200 mV and -400 mV,

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“…In 1951 Theorell and Chance [30] recognized the mechanistic importance of binary complex formation between enzyme and coenzyme in the sequence of kinetic steps during LADH catalysis. They demonstrated that this compulsory step precedes the formation of the productive ternary complex in the direction of alcohol oxidation, thus leading to an ordered, sequential pathway.…”

Section: General Importance Of Conformational Equilibriamentioning

confidence: 99%

The binding of 1,10‐phenanthroline to specifically active‐site cobalt(II)‐substituted horse‐liver alcohol dehydrogenase

Helander

Lindner

Brade

et al. 1988

European Journal of Biochemistry

202

140

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We have studied the binding of 1 ,lo-phenanthroline to specifically active-site cobalt(I1)-substituted horse-liver alcohol dehydrogenase [Co(II)-LADH]. The dissociation constant is a factor of 6500 smaller than in the native enzyme. Spectral evidence is given which shows that 1 ,lo-phenanthroline does not remove the catalytic Co(I1) ion and that binding of 1 ,I 0-phenanthroline renders the catalytic metal ion pentacoordinate. The maximum limiting rate constant for the association of 1,lO-phenanthroline to Co(I1)-LADH is about 60 s -' . This is about a third of the value (1 69 s-') determined for native horse-liver alcohol dehydrogenase, Zn(1I)LADH [Frolich et al. (1978) Arch. Biochem. Biuphys. 189,471 -4801. For cadmium(l1)-substituted horse-liver alcohol dehydrogenase, [Cd(II)LADH] the maximum limiting rate constant for association of 1 ,lo-phenanthroline increased to 590 s-'. These findings demonstrate that the rate-limiting step is strongly dependent on the chemical nature of the catalytic metal ion and its immediate environment. 1,lO-Phenanthroline is shown to bind to the Co(I1)-LADH . NAD' complex in the open conformation. The maximum limiting rate constant remains unchanged in the presence of NAD'. The data have been used to derive a kinetic scheme for the formation of ternary complexes including NAD' that involves a slow intermediary step.

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Studies on Liver Alcohol Dehydrogenase. II. The Kinetics of the Compound of Horse Liver Alcohol Dehydrogenase and Reduced Diphosphopyridine Nucleotide.

Cited by 448 publications

References 0 publications

Kinetic studies of formate dehydrogenase

Kinetic studies of formate dehydrogenase

Kinetic Studies of a Soluble αβ Complex of Nitrate Reductase A from Escherichia Coli

The binding of 1,10‐phenanthroline to specifically active‐site cobalt(II)‐substituted horse‐liver alcohol dehydrogenase

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