A product-inhibition study of bovine liver glutamate dehydrogenase

P C Engel; S S Chen

doi:10.1042/bj1510305

A product-inhibition study of bovine liver glutamate dehydrogenase

Biochemical Journal ◽

10.1042/bj1510305 ◽

1975 ◽

Vol 151 (2) ◽

pp. 305-318 ◽

Cited By ~ 25

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.

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Purification and properties of the soluble carnitine palmitoyltransferase from bovine liver mitochondria

Biochemical Journal ◽

10.1042/bj2440271 ◽

1987 ◽

Vol 244 (2) ◽

pp. 271-278 ◽

Cited By ~ 26

Author(s):

R R Ramsay ◽

J P Derrick ◽

A S Friend ◽

P K Tubbs

Keyword(s):

Glutamate Dehydrogenase ◽

Gel Filtration ◽

Random Order ◽

Bovine Liver ◽

Liver Mitochondria ◽

Carnitine Palmitoyltransferase ◽

Purification And Properties ◽

Rapid Equilibrium ◽

Variable Substrate ◽

Myristoyl Coa

A new carnitine palmitoyltransferase (CPT) was purified to homogeneity from bovine liver mitochondria which were 96% free of peroxisomal contamination, as judged by catalase and glutamate dehydrogenase activities. The enzyme is easily removed from mitochondria, without the use of detergent. It is monomeric (Mr 63,500), unlike other preparations of CPT from mitochondria, and is most active with myristoyl-CoA and palmitoyl-CoA. The Km values are between 0.8 and 4 microM for a range of substrates from hexanoyl-CoA to stearoyl-CoA; these are much lower than values reported for other purified CPT preparations. The Km for L-carnitine is 185 microM measured with palmitoyl-CoA, and does not vary greatly with the chain length. This is also lower than the values reported for other CPT preparations, but higher than those cited for the medium-chain transferases. Kinetic and inhibitor studies were consistent with a rapid-equilibrium random-order mechanism. 2-Bromopalmitoyl-CoA, which is an inhibitor of the outer CPT, inhibited the enzyme competitively with palmitoyl-CoA as the variable substrate, when added without preincubation. If the enzyme was preincubated with 2-bromopalmitoyl-CoA and carnitine, the activity did not reappear after gel filtration of the protein. The inhibitor was bound in a 1:1 stoichiometry per subunit of enzyme.

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

Biochemical Journal ◽

10.1042/bj2110099 ◽

1983 ◽

Vol 211 (1) ◽

pp. 99-107 ◽

Cited By ~ 6

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.

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

Biochemical Journal ◽

10.1042/bj1260975 ◽

1972 ◽

Vol 126 (4) ◽

pp. 975-984 ◽

Cited By ~ 69

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.

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

Biochemical Journal ◽

10.1042/bj1180409 ◽

1970 ◽

Vol 118 (3) ◽

pp. 409-419 ◽

Cited By ~ 62

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.

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Kinetic studies on the two common inherited forms of human erythrocyte adenylate kinase

Biochemical Journal ◽

10.1042/bj1300805 ◽

1972 ◽

Vol 130 (3) ◽

pp. 805-811 ◽

Cited By ~ 7

Author(s):

C. Brownson ◽

N. Spencer

Keyword(s):

Human Erythrocyte ◽

Adenylate Kinase ◽

Competitive Inhibition ◽

Kinetic Studies ◽

Forward Direction ◽

Product Inhibition ◽

Reverse Direction ◽

Kinetic Properties ◽

Ping Pong ◽

Variable Substrate

1. The kinetic properties of two genetic variants of human erythrocyte adenylate kinase were studied at limiting concentrations of both ADP and MgADP- in the forward direction and at limiting concentrations of both AMP and MgATP2- in the reverse direction. 2. Primary reciprocal plots rule out the possibility of a Ping Pong mechanism for both forms of the enzyme. 3. Analysis of the kinetic data by an appropriate computer program gave the following Km values for the type 1 enzyme: AMP, 0.33mm±0.1; MgATP2-, 0.95mm±0.13; ADP, 0.12mm±0.03; MgADP-, 0.22mm±0.04. Values for the type 2 enzyme were: AMP, 0.27mm±0.03; MgATP2-, 0.40mm±0.05; ADP, 0.08mm±0.07; MgADP-, 0.20mm±0.04. 4. Product inhibition studies were done by studying the reverse reaction. With ADP as product inhibitor competitive inhibition patterns were obtained with AMP and/or MgATP2- as variable substrate. Similar results were obtained for product inhibition by MgADP- with AMP as variable substrate. The results are consistent with a Rapid Equilibrium Random mechanism. 5. Secondary plots of slope versus product concentration were linear. The data were fitted to the appropriate equation and analysed by computer to give values for the product inhibition constants. 6. Differences between the values of certain kinetic constants for the two forms of the enzyme were observed.

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The mechanism of the reaction catalysed by adenosine triphosphate-creatine phosphotransferase

Biochemical Journal ◽

10.1042/bj0970037 ◽

1965 ◽

Vol 97 (1) ◽

pp. 37-52 ◽

Cited By ~ 227

Author(s):

JF Morrison ◽

E James

Keyword(s):

Initial Velocity ◽

Dissociation Constants ◽

Product Inhibition ◽

Ternary Complexes ◽

The Other ◽

Velocity Pattern ◽

Dead End ◽

Rapid Equilibrium ◽

Inhibition Pattern ◽

Mechanism Of The Reaction

1. The forward and reverse reactions catalysed by ATP-creatine phosphotransferase have been studied kinetically at pH8.0 in the presence and absence of products, under conditions in which the free Mg(2+) concentration was maintained constant at 1mm. Thus at fixed pH the reaction may be considered as being bireactant and expressed as:MgATP(2-)+creatine(0)right harpoon over left harpoonMgADP(-)+phosphocreatine(2-)2. The initial-velocity pattern in the absence of products and the product-inhibition pattern have been determined. These are consistent with a random mechanism in which all steps are in rapid equilibrium except that concerned with the interconversion of the central ternary complexes, and in which two dead-end complexes (enzyme-MgADP-creatine and enzyme-MgATP-phosphocreatine) are formed. The results are in accord with previous suggestions that the enzyme possesses distinct sites for the combination of the nucleotide and guanidino substrates. 3. Values have been determined for the Michaelis and dissociation constants involved in the combination of each substrate with various enzyme forms. Although these values cannot be regarded as absolute, they appear to indicate that the presence of one substrate on the enzyme enhances the combination of the second substrate. In addition, it would seem that in the formation of the enzyme-MgADP-creatine complex the concentration of one reactant does not affect the combination of the other. This contrasts with the formation of the enzyme-MgATP-phosphocreatine complex, where each reactant hinders the combination of the other.

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Synthesis of 5-Substituted Isophthalic Acids and Competitive Inhibition Studies with Bovine Liver Glutamate Dehydrogenase

Experimental Biology and Medicine ◽

10.3181/00379727-151-39200 ◽

1976 ◽

Vol 151 (2) ◽

pp. 316-320 ◽

Cited By ~ 6

Author(s):

S. G. Boots ◽

M. A. Franklin ◽

B. Dunlavey ◽

J. Costello ◽

C. Lipsitz ◽

...

Keyword(s):

Glutamate Dehydrogenase ◽

Competitive Inhibition ◽

Bovine Liver ◽

Inhibition Studies

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Ox liver glutamate dehydrogenase. The role of lysine-126 reappraised in the light of studies of inhibition and inactivation by pyridoxal 5'-phosphate

Biochemical Journal ◽

10.1042/bj1490619 ◽

1975 ◽

Vol 149 (3) ◽

pp. 619-626 ◽

Cited By ~ 13

Author(s):

S S Chen ◽

P C Engel

Keyword(s):

Glutamate Dehydrogenase ◽

Time Course ◽

Competitive Inhibition ◽

Bovine Liver ◽

Chemical Modifier ◽

Order Analysis ◽

The One ◽

Step Mechanism

The time-course of inactivation of bovine liver glutamate dehydrogenase by pyridoxal 5'-phosphate was studied in the presence of varied amounts of 2-oxoglutarate or NADH. Pseudo-first-order analysis reveals that the protection by both these compounds is competitive with respect to the chemical modifier. The competition is only partial, however: saturation with either NADH or 2-oxoglutarate decreases the rate constant for inactivation to a finite minimum and not to zero. Similarly, the plot of activity at equilibrium as a function of the concentration of the protecting substrate or coenzyme reveals that neither NADH nor 2-oxoglutarate protects completely against inactivation. In initial-rate experiments, pyridoxal 5'-phosphate, used as an instantaneous inhibitor rather than a long-term inactivator, displayed non-competitive inhibition with respect to both 2-oxoglutarate and NADH. These results clearly indicate that, although there is mutual hindrance between the binding to the enzyme of pyridoxal 5'-phosphate, on the one hand, and 2-oxoglutarate or NADH on the other, binding is not mutually exclusive. These findings are discussed in terms of the two-step mechanism for inactivation by pyridoxal 5'-phosphate. It is concluded that lysine-126 cannot be solely responsible for binding either the substrate or the coenzyme, but could be essential for the catalytic step.

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