scholarly journals Kinetic studies of glutamate dehydrogenase. The reductive amination of 2-oxoglutarate

1970 ◽  
Vol 118 (3) ◽  
pp. 409-419 ◽  
Author(s):  
P. C. Engel ◽  
K. Dalziel
Keyword(s):  
Glutamate Dehydrogenase ◽  
Reductive Amination ◽  
Initial Rate ◽  
Dissociation Constants ◽  
Kinetic Studies ◽  
Random Order ◽  
Order Type ◽  
Reverse Reaction ◽  
Enzyme Subunits ◽  
High Concentrations

1. Kinetic studies of the reductive amination of 2-oxoglutarate catalysed by glutamate dehydrogenase with NADH and NADPH as coenzyme were made at pH7.0 and pH 8.0. The concentrations of both substrates and coenzymes were simultaneously varied over wide ranges. Lineweaver–Burk plots with respect to each substrate and coenzyme were linear, except that with high concentrations of 2-oxoglutarate or coenzyme inhibition occurred. There was no evidence of the negative homotropic interactions between the enzyme subunits that were revealed in previous kinetic studies of the reverse reaction. 2. The initial-rate results are shown to be inconsistent with any of the six possible compulsory-order mechanisms for this three-substrate reaction, and it is concluded that a random-order mechanism is the most likely one. On the basis of this mechanism, the dissociation constants of all the binary, ternary and quaternary complexes of the enzyme and substrates are calculated from initial-rate parameters. 3. The results are discussed in relation to those of earlier workers who concluded that the mechanism is of the compulsory-order type.

scholarly journals The binding of oxidized coenzymes by glutamate dehydrogenase and the effects of glutarate and purine nucleotides

1972 ◽  
Vol 126 (4) ◽  
pp. 975-984 ◽  
Author(s):  
K. Dalziel ◽  
R. R. Egan
Keyword(s):  
Glutamate Dehydrogenase ◽  
Dissociation Constants ◽  
Equilibrium Dialysis ◽  
Kinetic Studies ◽  
Binding Studies ◽  
Purine Nucleotides ◽  
Active Centre ◽  
Sodium Phosphate Buffer ◽  
Low Concentrations ◽  
High Concentrations

1. The binding of NAD+ and NADP+ to glutamate dehydrogenase has been studied in sodium phosphate buffer, pH7.0, by equilibrium dialysis. Approximate values for the dissociation constants are 0.47 and 2.5mm respectively. For NAD+ the value agrees with that estimated from initial-rate results. 2. In the presence of the substrate analogue glutarate both coenzymes are bound more firmly, and there is one active centre per enzyme subunit. The binding results cannot be described in terms of independent and identical active centres, and binding is stronger at low coenzyme concentrations than at high concentrations. Either the six subunits of the oligomer are not identical or there are negative interactions between them in the binding of coenzymes in ternary complexes with glutarate. The latter explanation is favoured. 3. The binding studies support the conclusions drawn from earlier kinetic studies of the glutamate reaction. 4. ADP and GTP respectively decrease and increase the affinity of the enzyme for NAD+ and NADP+, in both the presence and absence of glutarate. The negative binding interactions in the presence of glutarate are abolished by ADP, which decreases the affinity for the coenzymes at low concentrations of the latter. 5. In the presence of glutarate, GTP and NAD+ or NADP+, the association of enzyme oligomers is prevented, and the solubility of the enzyme is decreased; the complex of enzyme and ligands readily crystallizes. 6. The results are discussed in relation to earlier kinetic studies.

scholarly journals Kinetics and reaction mechanism of yeast alcohol dehydrogenase with long-chain primary alcohols

1976 ◽  
Vol 157 (1) ◽  
pp. 15-22 ◽  
Author(s):  
W Schöpp ◽  
H Aurich
Keyword(s):  
Alcohol Dehydrogenase ◽  
Dissociation Constants ◽  
Kinetic Studies ◽  
Random Order ◽  
Primary Alcohols ◽  
Yeast Alcohol Dehydrogenase ◽  
Kinetic Coefficients ◽  
Non Linear ◽  
High Concentrations ◽  
Long Chain

Kinetic studies of yeast alcohol dehydrogenase with NAD+ and ethanol, hexanol or decanol as substrates invariably result in non-linear Lineweaver-Burk plots if the alcohol is the variable substrate. The kinetic coefficients determined from secondary plots are consistent with an ‘equilibrium random-order‘ mechanism for extremely low alcohol concentrations and for all alcohols, the transformation of the ternary complexes being the rate-limiting step of the reaction. This mechanism also applies to long-chain substrates at high concentrations, whereas the rate of the ethanol-NAD+ reaction at high ethanol concentrations is determined by the dissociation of the enzyme-NADH complex. The dissociation constants for the enzyme-NAD+ complex and for the enzyme-alcohol complexes obtained from the kinetic quotients satisfactorily correspond to the dissociation constants obtained by use of other techniques. It is suggested that the non-linear curves may be attributed to a structural change in the enzyme itself, caused by the alcohol.

scholarly journals Kinetic studies of dogfish liver glutamate dehydrogenase

1979 ◽  
Vol 177 (2) ◽  
pp. 449-459 ◽  
Author(s):  
A H Electricwala ◽  
F M Dickinson
Keyword(s):  
Glutamate Dehydrogenase ◽  
Kinetic Data ◽  
Reductive Amination ◽  
Initial Rate ◽  
Kinetic Studies ◽  
Bovine Liver ◽  
Binding Studies ◽  
Degree Of Protection ◽  
Binding Sequence ◽  
Kinetic Features

Initial-rate studies were made of the oxidation of L-glutamate by NAD+ and NADP+ catalysed by highly purified preparations of dogfish liver glutamate dehydrogenase. With NAD+ as coenzyme the kinetics show the same features of coenzyme activation as seen with the bovine liver enzyme [Engel & Dalziel (1969) Biochem. J. 115, 621–631]. With NADP+ as coenzyme, initial rates are much slower than with NAD+, and Lineweaver–Burk plots are linear over extended ranges of substrate and coenzyme concentration. Stopped-flow studies with NADP+ as coenzyme give no evidence for the accumulation of significant concentrations of NADPH-containing complexes with the enzyme in the steady state. Protection studies against inactivation by pyridoxal 5′-phosphate indicate that NAD+ and NADP+ give the same degree of protection in the presence of sodium glutarate. The results are used to deduce information about the mechanism of glutamate oxidation by the enzyme. Initial-rate studies of the reductive amination of 2-oxoglutarate by NADH and NADPH catalysed by dogfish liver glutamate dehydrogenase showed that the kinetic features of the reaction are very similar with both coenzymes, but reactions with NADH are much faster. The data show that a number of possible mechanisms for the reaction may be discarded, including the compulsory mechanism (previously proposed for the enzyme) in which the sequence of binding is NAD(P)H, NH4+ and 2-oxoglutarate. The kinetic data suggest either a rapid-equilibrium random mechanism or the compulsory mechanism with the binding sequence NH4+, NAD(P)H, 2-oxoglutarate. However, binding studies and protection studies indicate that coenzyme and 2-oxoglutarate do bind to the free enzyme.

scholarly journals Kinetic studies of glutamate dehydrogenase with glutamate and norvaline as substrates. Coenzyme activation and negative homotropic interactions in allosteric enzymes

1969 ◽  
Vol 115 (4) ◽  
pp. 621-631 ◽  
Author(s):  
P. C. Engel ◽  
K. Dalziel
Keyword(s):  
Glutamate Dehydrogenase ◽  
Phosphate Buffer ◽  
Initial Rate ◽  
Kinetic Studies ◽  
Glutamate Concentration ◽  
Enzyme Subunits ◽  
New Type ◽  
Michaelis Constants ◽  
Allosteric Enzymes ◽  
Absence Of Evidence

1. Kinetic studies of glutamate dehydrogenase were made with wide concentration ranges of the coenzymes NAD+ and NADP+ and the substrates glutamate and norvaline. Initial-rate parameters were evaluated. 2. Deviations from Michaelis–Menten behaviour towards higher activity were observed with increasing concentrations of either coenzyme with glutamate as substrate, but not with norvaline as substrate. 3. In phosphate buffer, pH7·0, Lineweaver–Burk plots with either coenzyme as variable and a constant, large glutamate concentration showed three or four linear regions of different slope with relatively sharp discontinuities. Maximum rates obtained by extrapolation and Michaelis constants for the coenzymes increased in steps with increase of coenzyme concentration. 4. In the absence of evidence of heterogeneity of the enzyme and coenzyme preparations, the results are interpreted in terms of negative homotropic interactions between the enzyme subunits. It is suggested that sharp discontinuities in Lineweaver–Burk plots or reciprocal binding plots may be characteristic of this new type of interaction, which can be explained in terms of an Adair–Koshland model, but not by the model of Monod, Wyman & Changeux.

scholarly journals Kinetic studies of the mechanism of pig kidney aldehyde reductase

1981 ◽  
Vol 193 (2) ◽  
pp. 485-492 ◽  
Author(s):  
F F Morpeth ◽  
F M Dickinson
Keyword(s):  
Initial Rate ◽  
Kinetic Studies ◽  
Random Order ◽  
Alcohol Oxidation ◽  
Aldehyde Reductase ◽  
Pig Kidney ◽  
Binary Complexes ◽  
Inhibition Studies ◽  
Kinetics Of ◽  
Aldehyde Reduction

Initial-rate measurements were made of the oxidations of pyridine-3-methanol and glycerol by NADP+ and of the reduction of the corresponding aldehydes by NADPH catalysed by pig kidney aldehyde reductase. In addition, a brief survey of the specificity of the enzyme towards aldehyde substrates and its sensitivity to the inhibitors ethacrynic acid, sodium barbitone and warfarin was made. The detailed kinetic work indicates a compulsory mechanism for aldehyde reduction, with NADPH binding before aldehyde. For alcohol oxidation, however, it is necessary to postulate the formation of kinetically significant amounts of binary complexes of the type enzyme-alcohol to explain the results. Thus, for alcohol oxidation random-order addition of substrates may occur. Inhibition studies of the kinetics of aldehyde reduction in the presence of the corresponding alcohol product provide further evidence for the existence of enzyme-alcohol complexes. Finally, detailed kinetic studies were made of the inhibition of pyridine-3-aldehyde reduction by sodium barbitone. The mechanism of the inhibition is discussed.

scholarly journals A steady-state random-order mechanism for the oxidative deamination of norvaline by glutamate dehydrogenase

1983 ◽  
Vol 211 (1) ◽  
pp. 99-107 ◽  
Author(s):  
C LiMuti ◽  
J E Bell
Keyword(s):  
Steady State ◽  
Glutamate Dehydrogenase ◽  
Dissociation Constants ◽  
Random Order ◽  
Kinetic Mechanism ◽  
Order Model ◽  
Oxidative Deamination ◽  
Steady State Kinetics ◽  
Rapid Equilibrium ◽  
Good Agreement

The kinetic mechanism of glutamate dehydrogenase with the monocarboxylic substrate norvaline was examined by using initial-rate steady-state kinetics and inhibition kinetics. To a first approximation the reaction mechanism can be described as a rapid-equilibrium random-order one. Binding synergism between the monocarboxylic substrate and coenzyme is not observed. Dissociation constants for NAD+ and 2-oxoglutarate calculated from the kinetic data assuming a rapid-equilibrium random-order model are in good agreement with independently obtained estimates. Lineweaver-Burk plots with varied norvaline concentration are not strictly linear, and it is concluded that a steady-state random-order model more accurately reflects the observed kinetics with norvaline as substrate.

scholarly journals Estimation of rate dissociation constants involving ternary complexes in reactions catalysed by yeast alcohol dehydrogenase

1978 ◽  
Vol 171 (3) ◽  
pp. 629-637 ◽  
Author(s):  
F. Mark Dickinson ◽  
Christopher J. Dickenson
Keyword(s):  
Alcohol Dehydrogenase ◽  
Catalytic Mechanism ◽  
Dissociation Constants ◽  
Kinetic Studies ◽  
Product Inhibition ◽  
Stopped Flow ◽  
Phenol Red ◽  
Yeast Alcohol Dehydrogenase ◽  
Fluorescence Measurements ◽  
High Concentrations

Stopped-flow studies of oxidation of butan-1-ol and propan-2-ol by NAD+ in the presence of Phenol Red and large concentrations of yeast alcohol dehydrogenase give no evidence for the participation of a group of pKa approx. 7.6 in alcohol binding. Such a group has been implicated in ethanol binding to horse liver alcohol dehydrogenase [Shore, Gutfreund, Brooks, Santiago & Santiago (1974) Biochemistry13, 4185–4190]. The present result supports previous findings based on steady-state kinetic studies with the yeast enzyme. Stopped-flow studies of the yeast alcohol dehydrogenase-catalysed reduction of acetaldehyde by NADH in the presence of ethanol as product inhibitor indicate that the rate-limiting step is NAD+ release from the enzyme–NAD+–ethanol product complex. This finding permits calculation of K3, the dissociation constant for ethanol from the enzyme–NAD+–ethanol complex, by using the product-inhibition data of Dickenson & Dickinson (1978) (Biochem. J.171, 613–627). The calculations show that K3 varies very little with pH in the range 5.95–8.9, and this agrees with the findings of the stopped-flow experiments described above. Absorption and fluorescence measurements on mixtures of substrates and coenzymes in the presence of high concentrations of alcohol dehydrogenase have been used to estimate values for the ratio [enzyme–NADH–acetaldehyde]/ [enzyme–NAD+–ethanol] at equilibrium. The values obtained were in the range 0.11±0.04, and this value together with estimates of K3 was used to provide estimates of values for rate constants and dissociation constants for steps within the catalytic mechanism.

scholarly journals Negative co-operativity in glutamate dehydrogenase. Involvement of the 2-position in glutamate in the induction of conformational changes

1985 ◽  
Vol 225 (1) ◽  
pp. 209-217 ◽  
Author(s):  
E T Bell ◽  
C LiMuti ◽  
C L Renz ◽  
J E Bell
Keyword(s):  
Glutamate Dehydrogenase ◽  
Conformational Changes ◽  
Active Site ◽  
Dissociation Constants ◽  
Kinetic Studies ◽  
Fluorescence Properties ◽  
Subunit Interactions ◽  
Oxidative Deamination ◽  
Substrate Analogues

The 2-position substituent on substrates or substrate analogues for glutamate dehydrogenase is shown to be intimately involved in the induction of conformational changes between subunits in the hexamer by coenzyme. These conformational changes are associated with the negative co-operativity exhibited by this enzyme. 2-Oxoglutarate and L-2-hydroxyglutarate induce indications of co-operativity similar to those induced by the substrate of oxidative deamination, glutamate, in kinetic studies. Glutarate (2-position CH2) does not. A comparison of the effects of L-2-hydroxyglutarate and D-2-hydroxyglutarate or D-glutamate indicates that the 2-position substituent must be in the L-configuration for these conformational changes to be triggered. In addition, glutarate and L-glutamate in ternary enzyme-NAD(P)H-substrate complexes induce very different coenzyme fluorescence properties, showing that glutamate induces a different conformation of the enzyme-coenzyme complex from that induced by glutarate. Although glutamate and glutarate both tighten the binding of reduced coenzyme to the active site, the effect is much greater with glutamate, and the binding is described by two dissociation constants when glutamate is present. The data suggest that the two carboxy groups on the substrate are required to allow synergistic binding of coenzyme and substrate to the active site, but that interactions between the 2-position on the substrate and the enzyme trigger the conformational changes that result in subunit-subunit interactions and in the catalytic co-operativity exhibited by this enzyme.

scholarly journals A product-inhibition study of bovine liver glutamate dehydrogenase

1975 ◽  
Vol 151 (2) ◽  
pp. 305-318 ◽  
Author(s):  
P C Engel ◽  
S S Chen
Keyword(s):  
Glutamate Dehydrogenase ◽  
Competitive Inhibition ◽  
Dissociation Constants ◽  
Random Order ◽  
Bovine Liver ◽  
Product Inhibition ◽  
Rate Equations ◽  
Sodium Phosphate Buffer ◽  
Rapid Equilibrium ◽  
Variable Substrate

1. Initial rates of oxidative deamination of L-glutamate with NAD+ as coenzyme, and of reductive aminiation of 2-oxoglutarate with NADH as coenzyme, catalysed by bovine liver glutamate dehydrogenase were measured in 0.111 M-sodium phosphate buffer, pH 7, at 25 degrees C, in the absence and presence of product inhibitors. All 12 possible combinations of variable substrate and product inhibitor were used. 2. Strict competition was observed between NAD+ and NADH, and between glutamate and 2-oxoglutarate. All other inhibition patterns were clearly non-competitive, except for inhibition by NH4+ with NAD+ as variable substrate. Here the extrapolation did not permit a clear distinction between competitive and non-competitive inhibition. 3. Mutually non-competitive behaviour between glutamate and NH4+ indicates that these substrates can be bound at the active site simultaneously. 4. Primary Lineweaver-Burk plots and derived secondary plots of slopes and intercepts against inhibitor concentration were linear, with one exception: with 2-oxoglutarate as variable substrate, the replot of primary intercepts against inhibitory NAD+ concentration was curved. 5. Separate Ki values were evaluated for the effect of each product inhibitor on the individual terms in the reciprocal initial-rate equations. With this information it is possible to calculate rates for any combination of substrate concentrations within the experimental range with any concentration of a single product inhibitor. 6. The inhibition patterns are consistent with neither a simple compulsory-order mechanism nor a rapid-equilibrium random-order mechanism without modification. They can, however, be reconciled with either type of mechanism by postulating appropirate abortive complexes. Of the two compulsory sequences that have been proposed, one, that in which the order of binding is NADH, NH4+, 2-oxoglutarate, requires an implausible pattern of abortive complex-formation to account for the results. 7. On the basis of a rapid-equilibrium random-order mechanism, dissociation constants can be calculated from the Ki values. Where these can be compared with independent estimates from the kinetics of the uninhibited reaction or from direct measurements of substrate binding, the agreement is reasonable good. On balance, therefore, the results provide further support for the rapid-equilibrium random-order mechanism under these conditions.

scholarly journals The kinetic mechanism of ox liver glutamate dehydrogenase in the presence of the allosteric effector ADP. The oxidative deamination of L-glutamate

1984 ◽  
Vol 223 (1) ◽  
pp. 161-168 ◽  
Author(s):  
D P Hornby ◽  
M J Aitchison ◽  
P C Engel
Keyword(s):  
Glutamate Dehydrogenase ◽  
Initial Rate ◽  
Potassium Phosphate ◽  
Kinetic Studies ◽  
Kinetic Mechanism ◽  
Major Effect ◽  
Oxidative Deamination ◽  
Allosteric Effector ◽  
Steady State Kinetic ◽  
State Kinetic

In steady-state kinetic studies of ox liver glutamate dehydrogenase in 0.11 M-potassium phosphate buffer, pH7, at 25 degrees C, the concentration of ADP was varied from 0.5 to 1000 microM. Inhibition was observed except when the concentrations of both glutamate and coenzyme were high, when activation was seen. With NAD+ or NADP+ as coenzyme, 200 microM-ADP was sufficient to saturate the enzyme with respect to the major effect of this nucleotide. In the presence of 210 microM-ADP, widely varied concentrations of coenzyme give linear Lineweaver-Burk plots, in marked contrast with results obtained previously for kinetics without ADP. This has allowed evaluation for the reaction with NAD+, NADP+ and acetylpyridine-adenine dinucleotide (315 microM-ADP in the last case) of all four initial rate parameters, i.e. the phi coefficients in the equation: (Formula: see text) where A is coenzyme and B is glutamate. The relative constancy of phi B and of phi AB/phi A with the different coenzymes point to a compulsory-order mechanism with glutamate as the leading substrate. This conclusion, though unexpected, agrees well with various previous observations on the binding of oxidized coenzyme.

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