The chymotrypsin-catalysed activation of bovine liver glutamate dehydrogenase.
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ABSTRACT:
SUBMITTER: Place GA
PROVIDER: S-EPMC1158448 | biostudies-other | 1982 Jul
REPOSITORIES: biostudies-other
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Project description: Not available
| EMPIAR-10217 | biostudies-other
Project description:In this work, we re-examine the previously reported phenomenon of the creation of a superactive glutamate dehydrogenase by proteolytic modification by chymotrypsin and explore the various discrepancies that came to light during those studies. We find that superactivation is caused by cleavage at the N terminus of the protein and not the C-terminal allosteric site, as has previously been suggested. N-terminal sequencing reveals that TLCK-treated chymotrypsin cleaves bovine glutamate dehydrogenase at phenylalanine 10. We suggest that trypsin contamination in nontreated chymotrypsin may have led to the production of the larger 4-5 kDa digestion product, previously misinterpreted as having caused the activation. In line with some previous studies, we can confirm that GTP inhibition is attenuated to some extent by the proteolysis, while ADP activation is almost abolished. Utilizing the recently solved structures of bovine glutamate dehydrogenase, we illustrate the cleavage points.
| S-EPMC2492812 | biostudies-literature
Project description: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.
| S-EPMC1172361 | biostudies-other
Project description:Bovine liver glutamate dehydrogenase is potently inhibited by Zn2+ ions. At pH 7.0 a kinetic dissociation constant for Zn2+ of 18 microM is obtained. The fluorescent lanthanide Eu3+ competes for the Zn2+-binding site and relieves the Zn2+-induced inhibition, but does not cause inhibition. Studies on the effects of Zn2+ or Eu3+ on the tertiary and quaternary structure of the enzyme by the use of protein fluorescence, heat-stability and re-activation after guanidinium chloride denaturation indicate that, whereas Zn2+ affects both tertiary and quaternary structure, Eu3+ does not affect either, consistent with its lack of effect on enzymic properties. Eu3+ fluorescence had a strong excitation peak at 395 nm with emission at 456 nm. In the presence of glutamate dehydrogenase the fluorescence emission is shifted to 501 nm. Eu3+, with high-affinity binding site and distinctive fluorescence properties after binding, would appear to be an ideal fluorophore for use in conformational studies or resonance-energy-transfer studies.
| S-EPMC1148258 | biostudies-other
Project description:Glutamate dehydrogenase (GDH) catalyzes reversible conversion between glutamate and 2-oxoglutarate using NAD(P)(H) as a coenzyme. Although mammalian GDH is regulated by GTP through the antenna domain, little is known about the mechanism of allosteric activation by leucine. An extremely thermophilic bacterium, Thermus thermophilus, possesses GDH with a unique subunit configuration composed of two different subunits, GdhA (regulatory subunit) and GdhB (catalytic subunit). T. thermophilus GDH is unique in that the enzyme is subject to allosteric activation by leucine. To elucidate the structural basis for leucine-induced allosteric activation of GDH, we determined the crystal structures of the GdhB-Glu and GdhA-GdhB-Leu complexes at 2.1 and 2.6 Å resolution, respectively. The GdhB-Glu complex is a hexamer that binds 12 glutamate molecules: six molecules are bound at the substrate-binding sites, and the remaining six are bound at subunit interfaces, each composed of three subunits. The GdhA-GdhB-Leu complex is crystallized as a heterohexamer composed of four GdhA subunits and two GdhB subunits. In this complex, six leucine molecules are bound at subunit interfaces identified as glutamate-binding sites in the GdhB-Glu complex. Consistent with the structure, replacement of the amino acid residues of T. thermophilus GDH responsible for leucine binding made T. thermophilus GDH insensitive to leucine. Equivalent amino acid replacement caused a similar loss of sensitivity to leucine in human GDH2, suggesting that human GDH2 also uses the same allosteric site for regulation by leucine.
| S-EPMC3199488 | biostudies-literature
Project description:The liver is the most essential organ for the metabolism of ammonia, in where most of ammonia is removed by urea and glutamine synthesis. Regulated by leucine, glutamate dehydrogenase (GDH) catalyzes the reversible inter-conversion of glutamate to ammonia. To determine the mechanism of leucine regulating GDH, pigs weighing 20 ± 1 kg were infused for 80 min with ammonium chloride or alanine in the presence or absence of leucine. Primary pig hepatocytes were incubated with or without leucine. In the in vivo experiments with either ammonium or alanine as the nitrogen source, addition of leucine significantly inhibited ureagenesis and promoted the production of glutamate and glutamine in the perfused pig liver (P < 0.05). Similarly, leucine stimulated GDH activity and inhibited sirtuin4 (SIRT4) gene expression (P < 0.01). Leucine could also activate mammalian target of rapamycin complex 1 (mTORC1) signaling (P < 0.05), as evidenced by the increased phosphorylation levels of ribosomal protein S6 kinase 1 (S6K1) and ribosomal protein S6 (S6). Interestingly, the leucine-induced mTORC1 pathway activation suitably correlated with increased GDH activity and decreased expression of SIRT4. Similar results were observed in primary cultured hepatocytes. Notably, leucine exerted no significant change in GDH activity in SIRT4-deficient hepatocytes (P > 0.05), while mTORC1 signaling was activated. Leucine exerted no significant changes in both GDH activity and SIRT4 gene expression in rapamycin treated hepatocytes (P > 0.05). In conclusion, L-leucine increases GDH activity and stimulates glutamate synthesis from different nitrogen sources by regulating mTORC1/SIRT4 pathway in the liver of pigs.
| S-EPMC6116330 | biostudies-literature
Project description:1. The activity of liver alcohol dehydrogenase with cyclohexanol and cyclohexanone as substrates was studied, and the initial-rate parameters were determined from measurements at low substrate concentrations. In contrast with aliphatic ketones, cyclohexanone is a fairly good substrate, although less active than aliphatic aldehydes. The Michaelis constant for cyclohexanol is of the same order as that for ethanol, and the maximum rate and Michaelis constant for NAD(+) obtained with cyclohexanol are very similar to those obtained with primary aliphatic alcohols. The data for this substrate at low concentrations are therefore consistent with a compulsory-order mechanism in which ternary complexes are not rate-limiting. 2. With large concentrations of NAD(+), substrate activation is observed with increasing concentrations of cyclohexanol, whereas with small NAD(+) concentrations substrate inhibition is observed. This complex behaviour is explained by a mechanism previously proposed for this enzyme, which also satisfactorily described the kinetics of oxidation of primary and secondary aliphatic alcohols and aldehydes, including the substrate inhibition exhibited by primary alcohols, and the reduction of aldehydes. The activation with large concentrations of both NAD(+) and cyclohexanol is attributed to the formation of an abortive complex, E.NADH.ROH, from which NADH dissociates more rapidly than from the normal product complex E.NADH. Substrate inhibition in the presence of small NAD(+) concentrations is attributed to the formation of an active complex E.ROH, with which NAD(+) reacts more slowly than with the free enzyme. 3. Some support for these mechanisms of substrate activation and inhibition is obtained by approximate theoretical calculations, and their applicability to other two-substrate reactions that exhibit complex initial-rate behaviour, as a more likely alternative to the postulate of a second binding site for the substrate, is suggested.
| S-EPMC1265162 | biostudies-other
Project description:1. Kinetic aspects of the reaction between crystalline bovine liver glutamate dehydrogenase and formiminoglutamate were investigated to establish the conditions under which the latter may interfere with the assay of glutamate by using glutamate dehydrogenase and to explain why formiminoglutamate accumulates in vivo after histidine loading, although it can react with glutamate dehydrogenase. The Km and Vmax. values were compared with those of the enzyme reacting with glutamate. At pH 7.4 Km for formiminoglutamate was much higher and Vmax. much lower than the values for glutamate. 2. The equilibrium constant at pH 7.0 was 0.017 micrometer with formiminoglutamate, i.e. about one two-hundredths that with glutamate. 3. In vivo the interaction between glutamate dehydrogenase and formiminoglutamate is minimal even when the concentration of the latter in the liver is greatly raised, as in cobalamine or folate deficiency after histidine loading. 4. At pH 9.3, i.e. under the conditions for the assay of glutamate by glutamate dehydrogenase, formiminoglutamate reacts readily with the enzyme.
| S-EPMC1183950 | biostudies-other
Project description: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.
| S-EPMC1186394 | biostudies-other
Project description:Previous workers have shown that the hexamers of glutamate dehydrogenase are dissociated first into trimers and subsequently into monomers by increasing guanidinium chloride concentrations. In renaturation experiments it is shown that trimers of glutamate dehydrogenase can be reassociated to give the hexamer form of the enzyme, with full regain of activity. Monomeric subunits produced at high guanidinium chloride concentrations cannot be renatured. The trimer form of the enzyme is shown to have no catalytic activity, although the hexamer form in guanidinium chloride has full activity.
| S-EPMC1153214 | biostudies-other