Studies of Glutamate Dehydrogenase

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1. Equilibrium dialyses have shown tighter binding of GTP to glutamate dehydrogenase in triethanolamine than in phosphate buffer, as characterized by dissociation constants of 35 pM and 325 pM, respectively. If competition between GTP and phosphate occurs, a dissociation constant of 13 mM can be calculated for the binding of phosphate. Binding of ADP, however, is not changed when phosphate buffer is replaced by triethanolamine buffer.2. The binding of GTP and ADP to the enzyme as well as their mutual interaction in the bound state has also been examined by equilibrium dialyses. Both nucleotides bind simultaneously but with increased dissociation constants. For this negative interaction of ADP and GTP, a coupling energy of A @ = + 3.3 kJ mol-' (polypeptide chain)-' can be calculated. The total number of sites occupied by the two effectors in the ternary complex is identical to the sum of binding sites for the two nucleotides in their respective binary complexes. Thus separate although interacting sites exist for ADP and GTP.3. Nucleotide binding within the ternary glutamate dehydrogenase-NADPH-GTP complex has been correlated with ligand-induced dissociation of the enzyme to the unimer (the functional subunit comprising six polypeptide chains). Equilibrium dialyses revealed curved Scatchard plots indicating positive cooperative formation of the ternary complex from both binary enzyme-nucleotide complexes. In addition to indicating decreased dissociation constants of the two nucleotides, the binding curves reflect the ligand-induced dissociation of the enzyme into the unimer. Sedimentation analyses of the latter process show close agreement between the degree of ternary complex formation and the extent of dissociation of the enzyme. Two defined states of the enzyme can be distinguished. One state exists as the binary enzyme . nucleotide complex with an unchanged association equilibrium; the other state is completely dissociated into unimers which are saturated with both nucleotides, GTP and NADPH. The exchange between these two states is slow.4. The influence of ADP on the ternary enzyme-NADPH-GTP complex has been studied with respect to both the nucleotide binding and to the association equilibrium of the enzyme. ADP strongly decreases the affinity of the enzyme for both NADPH and GTP and, in addition, restores the ability of the enzyme to associate. The latter process appears to be complete after two or three of the total of six ADP-binding sites per unimer are saturated. The quaternary complex, enzyme-NADPH-GTP-ADP, stabilizes the associated state of the enzyme. However, at high concentration of GTP and ADP, the dissociation constant for NADPH binding to the enzyme is the same as in the enzyme . NADPH binary complex. Thus the effects of two purine nucleotides cancel each other.The enzymatic activity of beef liver glutamate dehydrogenase is modulated by purine nucleotides in a complex fashion [5,6]. ADP is known to be an activator above pH 7 whereas GTP strongly inhibits the enzymatic reaction under This work has...

Section: The Quuternury System Glutamate Drhydrogenase Adp Gtp N Asupporting

confidence: 80%

Studies of Glutamate Dehydrogenase

Dieter

Koberstein

Sund

2005

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“…More recent circular dichroism studies in the presence of GTP provided strong evidence for a maximum of two NADH molecules bound per polypeptide chain which are characterized by two distinct Cotton effects of opposite sign and different magnitude [15,16]. I n addition, direct binding measurements with the ultracentrifuge in the absence of GTP clearly show the existence of two binding sites per polypeptide chain for both reduced coenzymes, NADH and NADPH [1,2,9]. At the first binding site no differences in the affinity for NADH and NADPH were observed, the second site however has an approximately ten-fold higher affinity for NADH than for NADPH.…”

Section: 13)mentioning

confidence: 98%

“…I n solution the oligomers exhibit an association-dissociation equilibrium with higher This paper is dedicated to Theodor Wieland on the occasion of his 60th birthday. This work has been described in a preliminary report [1,2] …”

mentioning

confidence: 99%

Studies of Glutamate Dehydrogenase

Koberstein

Sund

1973

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The detailed investigation of the binding of NAD(P)H to beef liver glutamate dehydrogenase in the presence of the effectors ADP and GTP shows that each oligomer (composed of six identical polypeptide chains) has a total of I8 nucleotide binding sites. They can be subdivided into three classes. A first set of six binding sites, the active sites, is occupied by NAD(P)H with nearly the same affinity for both coenzymes, and accompanied by negative cooperativity between the binding sites within the oligomer. The second set of six binding sites, the nonactive sites, binds the coenzyme less tightly than the first set and ADP competes with NAD(P)H for these sites. The third set of six binding sites, the GTP sites, is responsible for the GTP binding.ADP abolishes the negative cooperativity of the NAD(P)H binding to the active sites and causes a weaker binding of both reduced coenzymes to these sites ( K E .ADP, NAD(P)H = 70 pM for NADH and 64 pM for NADPH). Also the reduced coenzymes weaken the binding of ADP to the enzyme. The dissociation constant for the enzyme * ADP complex is found to be 17 pM in the presence and 3 pM in the absence of NAD(P)H. The results are interpreted on the basis of free energy conservation in internal equilibria. The coupling energy AE", was calculated to be about I kcal/mol.I n contrast to ADP/NAD(P)H, GTP and NAD(P)H enhance each other's binding to glutamate dehydrogenase causing positive cooperativity which is stronger in the case of NADH than of NADPH. The Hill coefficient is found to be 2.1 for NADH and 1.8 for NADPH. The nonactive sites are occupied by NADH in the presence of GTP with a dissociation constant of 23 $5 indicating a three-fold enhancement of the affinity of the enzyme for NADH in the presence of GTP. This is not the case with NADPH, where the dissociation constant is about 600 pM. This value is similar to the dissociation constant of the binary complex.The influence of glutamate on the enzyme * NAD(P)H binding is similar to the effect obtained in the presence of GTP; glutamate and GTP, however, are most likely bound to M e r e n t sites. Glutamate induces strong positive cooperativity of three of the active sites within the oligomer characterized by a Hill coefficient of 2.4 for both reduced coenzymes. The extrapolations yield dissociation constants of 2-3 pM for both reduced coenzymes. The nonactive sites are occupied with dissociation constants of 57 pM for NADH and 700 pM for NADPH. These data indicate that glutamate has only a small effect on the binding of the reduced coenzyme to the nonactive site.The functional unit of beef liver glutamate dehydrogenase, the oligomer, is composed of six polypeptide chains with a molecular weight of 56000 [4-61. I n solution the oligomers exhibit an association-dissociation equilibrium with higher This paper is dedicated to Theodor Wieland on the occasion of his 60th birthday. This work has been described in a preliminary report [1,2]

“…Since the kinetic data with [SINADPH shows that the enzyme has enhanced activity with ADP present, it is apparent that ADP must compete at the reduced coenzyme regulatory site rather than at the catalytic site. Indeed, ADP has been reported to compete for the adenyl portion of the reduced coenzyme regulatory site [31,32,[34][35][36].…”

Section: Discussionmentioning

confidence: 99%

Distance between the substrate and regulatory reduced coenzyme binding sites of bovine liver glutamate dehydrogenase by resonance energy transfer

Lark

Colman

1990

Bovine liver glutamate dehydrogenase is known to bind reduced coenzyme at two sites/subunit, one catalytic and one regulatory; ADP competes for the latter site. The enzyme is here shown to be catalytically active with the thionicotinamide analogue of NADPH ([SINADPH) [7], and calf brain tubulin [8]. Energy transfer between two chromophores is possible as long as the emission spectrum of the energy donor overlaps the absorption spectrum of the energy acceptor. When combined with the spectral properties of the donor and acceptor, the amount of quenching of the donor's fluorescence by the acceptor leads to information about the distance between the chromophores.The activity of bovine liver glutamate dehydrogenase is affected by purine nucleotides. Either NADPH or NADH is capable of binding to the catalytic site [9] and high concentrations of these coenzymes inhibit the enzyme by binding to a distinct reduced coenzyme regulatory site [lo]. ADP, which activates the enzyme, has two binding sites per subunit [Ill.