Inactivation of glutamate dehydrogenase and glutamate synthase from Bacillus megaterium by phenylglyoxal, butane-2,3-dione and pyridoxal 5'-phosphate.
ABSTRACT: Reaction of phenylglyoxal with glutamate dehydrogenase (EC 220.127.116.11), but not with glutamate synthase (EC 18.104.22.168), from Bacillus megaterium resulted in complete loss of enzyme activity. NADPH alone or together with 2-oxoglutarate provided substantial protection from inactivation by phenylglyoxal. Some 2mol of [14C]Phenylglyoxal was incorporated/mol of subunit of glutamate dehydrogenase. Addition of 1mM-NADPH decreased incorporation by 0.7mol. The Ki for phenylglyoxal was 6.7mM and Ks for competition with NADPH was 0.5mM. Complete inactivation of glutamate dehydrogenase by butane-2,3-dione was estimated by extrapolation to result from the loss of 3 of the 19 arginine residues/subunit. NADPH, but not NADH, provided almost complete protection against inactivation. Butane-2,3-dione had only a slight inactivating effect on glutamate synthase. The data suggest that an essential arginine residue may be involved in the binding of NADPH to glutamate dehydrogenase. The enzymes were inactivated by pyridoxal 5'-phosphate and this inactivation increased 3--4-fold in the borate buffer. NADPH completely prevented inactivation by pyridoxal 5'-phosphate.
Project description:The arginine-specific reagents phenylglyoxal and butane-2,3-dione inactivated goat liver aminoacetone synthase with pseudo-first-order kinetics, with the rate dependent on modifier concentration. Phenylglyoxal and butane-2,3-dione appeared to react with one arginine residue per enzyme molecule. The inactivated enzyme could be re-activated by Tris, suggesting additional evidence of modification of the arginine residue. Acetyl-CoA, one of the substrates, completely protected the enzyme from inactivation. Glycine gave partial protection. Protection by substrates against inactivation by phenylglyoxal and butane-2,3-dione suggested the presence of an essential arginine residue at the substrate-binding region. Experiments with [7-14C]phenylglyoxal in the presence of acetyl-CoA showed that only the arginine residue at the active site could be modified by phenylglyoxal. The stability of the enzyme is dependent on the presence of both EDTA and Mg2+.
Project description:The effect of pyridoxal 5'-phosphate on the activity of ox liver glutamate dehydrogenase towards different amino acid substrates was investigated. Both alanine and glutamate activities decreased steadily in the presence of pyridoxal 5'-phosphate. The alanine/glutamate activity ratio increased as a function of inactivation by pyridoxal 5'-phosphate, indicating that glutamate activity is lost more rapidly than alanine activity. A mixture of NADH, GTP and 2-oxoglutarate completely protected the alanine and glutamate activities against inactivation by pyridoxal 5'-phosphate. The activity of glutamate dehydrogenase towards glutamate and leucine decreased steadily in a constant ratio in the presence of pyridoxal 5'-phosphate. The effect of leucine on the alanine and glutamate activities as a function of inactivation by pyridoxal 5'-phosphate was studied. The results are interpreted to suggest that the subunits of glutamate dehydrogenase hexamer are kinetically non-equivalent with regard to activity towards the two monocarboxylic amino acids as well as glutamate, and that all three substrates share the same active centre. However, leucine is also able to bind at a separate regulatory site.
Project description:Butane-2,3-dione inhibits the enzymic activity of Streptomyces griseus photoreactivating enzyme (PRE). Some characteristics of the inhibition, notably the enhancement by borate buffer and the reversibility, indicate that arginine residues are modified. From the kinetics of inhibition it can be concluded that a single essential arginine residue is involved. U.v.-irradiated DNA, the substrate for PRE, protects the enzyme against inactivation by butane-2,3-dione. This suggests that the essential arginine residue is situated in or near the u.v.-irradiated-DNA-binding site. Non-irradiated DNA at higher concentrations also protects against inactivation, indicating that PRE can form non-specific complexes. From the ratio of complex constants obtained from protection experiments with non-irradiated and u.v.-irradiated DNA it appears that PRE preferably binds to dimer sites.
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.
Project description:The arginine-specific reagent phenylglyoxal inactivated the active, dephosphorylated, form of Escherichia coli isocitrate dehydrogenase rapidly in a pseudo-first-order process. Both NADP+ and NADPH protected the enzyme against inactivation. Phenylglyoxal appeared to react with one arginine residue per subunit, and the extent of the reaction was proportional to the extent of the inactivation. In contrast, the phosphorylated form of isocitrate dehydrogenase did not react detectably with phenylglyoxal. The data indicate that the coenzyme-binding site of isocitrate dehydrogenase contains a reactive arginine residue that is protected by phosphorylation, and are consistent with the hypothesis that phosphorylation of the enzyme occurs close to or at its active site.
Project description:A kinetic analysis is presented of reactions of protein modification, and/or of modification-induced enzyme inactivation, which can formally be described by a single exponential function, or by a summation of two exponential functions, of reaction time plus a constant term. The reaction schemes compatible with the kinetic formalism of these cases are given, and a simple kinetic criterion is described whereby the identification of one of these cases, strong negative protein modification co-operativity, may be carried out. The treatment outlined in this paper is applied to a case from the literature, the inactivation of glyceraldehyde-3-phosphate dehydrogenase by butane-2,3-dione [Asriyants, Benkevich & Nagradova (1983) Biokhimiya (Engl. Transl.) 48, 164-171].
Project description:The presence of arginine at the active site of Leishmania donovani adenosine kinase was studied by chemical modification, followed by the characterization of the modified enzyme. The arginine-specific reagents phenylglyoxal (PGO), butane-2,3-dione and cyclohexane-1,2-dione all irreversibly inactivated the enzyme. In contrast, adenosine kinase from hamster liver was insensitive to these reagents. The inactivation of the enzyme by PGO followed pseudo-first-order kinetics, with a second-order rate constant of 39.2 min-1.M-1. Correlation between the stoichiometry of PGO modification and extent of inactivation indicated that modification of a single residue per molecule suffices for the loss of activity. Reactivity of the essential arginine residue towards PGO was affected by the presence of adenosine (Ado) and other competing alternative substrates, consistent with an arginine residue located proximal to the Ado-binding site. The enzyme showed an intrinsic fluorescence with an emission maximum at 340 nm when excited at 295 nm. The protein fluorescence was partially quenched on addition of Ado. PGO modification also led to significant quenching of the fluorescence. However, the fluorescence of the Ado-protected enzyme, which displayed 82% of the original activity after PGO treatment, was retained. The kinetic analyses of the partially modified enzyme showed an increase in the Km for Ado from 14 to 55 microM. Furthermore, the inability of the modified enzyme to bind to 5'-AMP-Sepharose 4B affinity column provided additional evidence that modification is attended by decrease in affinity of the enzyme for Ado. The results are consistent with the interpretation that modification of the active-site arginine residue affects activity by interfering with the binding of the substrate to the active site.
Project description:1. The activity of bovine liver glutamate dehydrogenase incubated with pyridoxal 5'-phosphate declined to a steady value reached within 30--60 min. The residual activity depended on the concentration of modifier up to about 5 mM. Above this concentration, however, no further inactivation was produced. The minimum activity obtainable in such incubations was 6--7% of the initial value. 2. Km values of the modified enzyme were unaltered, whereas Vmax. was decreased. 3. Activity was fully regained on dialysis against 0.1 M-potassium phosphate buffer. 4. Reduction with borohydride rendered the inactivation permanent but did not alter its extent. 5. Enzyme permanently inactivated in this way to the extent of 90% and dialysed was re-treated with pyridoxal 5'-phosphate. In this second cycle activity declined from 10 to 1% of the original activity. 6. This strongly suggests that the failure to achieve complete inactivation in a single cycle reflects a reversible equilibrium between inactive Schiff base, i.e. covalently modified enzyme, and a non-covalent complex. 7. The re-inactivation reaction occurring on dilution was demonstrated directly and a first-order rate constant obtained (0.048 min-1). This, in conjunction with an estimate of the forward rate constant for Schiff-base formation, obtained by approximate pseudo-first-order analysis of inactivation at varied modifier concentrations, gives a predicted minimum activity very close to that actually obtained in a single cycle of treatment. 8. The dissociation constant of the non-covalent complex is given by two methods as 0.90 and 1.59mM. 9. The results indicate that covalent modification with pyridoxal 5'-phosphate completely abolishes the activity of glutamate dehydrogenase.
Project description: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.
Project description:Bacillus megaterium N.C.T.C. no. 10342 exhibits glutamate synthetase (EC 22.214.171.124) and glutamate dehydrogenase (EC 126.96.36.199) activities. Concentrations of glutamate synthase were high when the bacteria were grown on 3mM-NH4Cl and low when they were grown on 100mM-NH4Cl, whereas glutamate dehydrogenase concentrations were higher when the bacteria were grown on 100mM-NH4Cl than on 3mM-NH4Cl. Glutamate synthase and glutamate dehydrogenase were purified to homogeneity from B. megaterium grown in 10mM-glucose/10mM-NH4Cl. The purified enzymes had mol.wts. 840000 and 270000 for glutamate synthase and glutamate dehydrogenase respectively. The Km values for substrates with NADPH and coenzyme were (glutamate synthase activity shown first) 9 micron and 360 micron for 2-oxoglutarate, 7.1 micron and 8.7 micron for NADPH, and 0.2 mM for glutamine and 22 mM for NH4Cl, similar values to those of enzymes from Escherichia coli. Glutamate synthase contained NH3-dependent activity (different from authentic glutamate dehydrogenase), which was enhanced 4-fold during treatment at pH 4.6 NH3-dependent activity was generally about 2% of the glutamine-dependent activity. Amidination of glutamate synthase by the bi-functional cross-linking reagent dimethyl suberimidate inactivated glutamine-dependent glutamate synthase activity, but increased NH3-dependent activity. A cross-linked structure of mol.wt. approx 200000 was the main product formed.