5'-p-fluorosulfonyl)benzoyl-8-azidoadenosine: a new bifunctional affinity label for nucleotide binding sites in proteins

A new bifunctional affinity label, 5'-p-(fluorosulfonyl)benzoyl-8-azidoadenosine (5'-FSBAzA), has been synthesized by condensation of p-(fluorosulfonyl)benzoyl chloride with 8-azidoadenosine. 5'-FSBAzA has been characterized by elemental analysis, thin-layer chromatography, and ultraviolet and 1H NMR spectroscopy. The affinity label contains both an electrophilic fluorosulfonyl moiety and a photoactivatable azido group which are capable of reacting with several classes of amino acids found in enzymes. 5'-FSBAzA reacts with bovine liver glutamate dehydrogenase in a two-step process: a dark reaction yielding about 0.5 mol of the sulfonylbenzoyl-8-azidoadenosine (SBAzA) group bound/mol enzyme subunit by reaction of the enzyme at the fluorosulfonyl group, followed by photolysis in which 25% of the covalently bound SBAzA becomes crosslinked to the enzyme. 5'-FSBAzA-modified glutamate dehydrogenase, both before and after photolysis, retains full catalytic activity but is less sensitive to allosteric inhibition by GTP, to activation by ADP, and to inhibition by 1 mM NADH. These results suggest the modification in the dark reaction of a regulatory nucleotide binding site. Photoactivation of the covalently bound reagent may have general applicability in relating modified amino acids which are close to each other in the region of the purine nucleotide binding sites of glutamate dehydrogenase and other proteins.     

Affinity labeling of bovine liver glutamate dehydrogenase with 8-[(4-bromo-2,3-dioxobutyl)thio]adenosine 5'-diphosphate and 5'-triphosphate

Bovine liver glutamate dehydrogenase reacts with 8-[(4-bromo-2,3-dioxobutyl)thio]adenosine 5'-diphosphate (8-BDB-TA-5'-DP) and 5'-triphosphate (8-BDB-TA-5'-TP) to yield enzyme with about 1 mol of reagent incorporated/mol of enzyme subunit. The modified enzyme is catalytically active but has decreased sensitivity to inhibition by GTP, reduced extent of activation by ADP, and diminished inhibition by high concentrations of NADH. Since modified enzyme, like native glutamate dehydrogenase, reversibly binds more than 1 mol each of ADP and GTP, it is unlikely that 8-BDB-TA-5'-TP reacts directly within either the ADP or GTP regulatory sites. The rate constant for reaction of enzyme exhibits a nonlinear dependence on reagent concentration with KD = 89 microM for 8-BDB-TA-5'-TP and 240 microM for 8-BDB-TA-5'-DP. The ligands ADP and GTP alone and NADH alone produce only small decreases in the rate constant for the reaction of enzyme with 8-BDB-TA-5'-TP, but the combined addition of 5 mM NADH + 200 microM GTP reduces the reaction rate constant more than 10-fold and the reagent incorporation to about 0.1 mol/mol of enzyme subunit. These results suggest that 8-BDB-TA-5'-TP reacts as a nucleotide affinity label in the region of the GTP-dependent NADH regulatory site of bovine liver glutamate dehydrogenase.     

Affinity labeling of an allosteric ADP site of glutamate dehydrogenase by 2-(4-bromo-2,3-dioxobutylthio)adenosine 5'-monophosphate

Bovine liver glutamate dehydrogenase reacts covalently with the adenine nucleotide analogue 2-(4-bromo-2,3-dioxobutylthio)adenosine 5'-monophosphate (2-BDB-TAMP) with incorporation of about 1 mol of reagent/mol of enzyme subunit. The modified enzyme is not inactivated by this reaction as measured in the absence of allosteric effectors. Native glutamate dehydrogenase is activated by ADP and inhibited by high concentrations of NADH; both of these effects are irreversibly decreased upon reaction of the enzyme with 2-BDB-TAMP. The decrease in activation by ADP was used to determine the rate constant for reaction with 2-BDB-TAMP. The rate constant (kobs) for loss of ADP activation exhibits a nonlinear dependence on 2-BDB-TAMP concentration, suggesting a reversible binding of reagent (KR = 0.74 mM) prior to irreversible modification. At 1.2 mM 2-BDB-TAMP, kobs = 0.060 min-1 and is not affected by alpha-ketoglutarate or GTP, but is decreased to 0.020 min-1 by 5 mM NADH and to zero by 5 mM ADP. Incorporation after incubation with 1.2 mM 2-BDB-TAMP for 1 h at pH 7.1 is 1.02 mol/mol enzyme subunit in the absence but only 0.09 mol/subunit in the presence of ADP. The enzyme protected with 5 mM ADP behaves like native enzyme in its activation by ADP and in its inhibition by NADH. Native enzyme binds reversibly 2 mol of [14C]ADP/subunit, whereas modified enzyme binds only 1 mol of ADP/peptide chain. These results indicate that incorporation of 1 mol of 2-BDB-TAMP causes elimination of one of the ADP sites of the native enzyme. 2-BDB-TAMP acts as an affinity label of an ADP site of glutamate dehydrogenase and indirectly influences the NADH inhibitory site.     

Distance relationships between the catalytic site labeled with 4-(iodoacetamido)salicylic acid and regulatory sites of glutamate dehydrogenase

The distance between the catalytic site on bovine liver glutamate dehydrogenase labeled with 4-(iodoacetamido)salicylic acid (ISA) and the adenosine 5'-diphosphate (ADP) activatory site occupied by the analogue 2',3'-O-(2,4,6-trinitrocyclohexadienylidene)adenosine 5'-diphosphate (TNP-ADP) was evaluated by energy transfer. Native enzyme and enzyme containing about 1 mol of acetamidosalicylate/mol of subunit bind about 0.5 mol of TNP-ADP/mol of subunit, and TNP-ADP competes for binding with ADP to native and modified enzyme, indicating that the analogue is a satisfactory probe of the ADP site. From the quenching of acetamidosalicylate donor fluorescence upon addition of TNP-ADP, an average distance of 33 A was determined between the catalytic and ADP sites. The fluorescent nucleotide analogue 5'-[p-(fluorosulfonyl)benzoyl]-2-aza-1,N6-ethenoadenosine (5'-FSBa epsilon A) reacts covalently with glutamate dehydrogenase to about 1 mol/peptide chain. As compared to native enzyme, the SBa epsilon A-enzyme exhibits decreased sensitivity to GTP inhibition but retains its catalytic activity as well as its ability to be activated by ADP and inhibited by high concentrations of NADH. Complete protection against decreased sensitivity to GTP inhibition is provided by GTP in the presence of NADH. It is concluded that 5'-FSBa epsilon A modifies a GTP site on glutamate dehydrogenase. The distance of 23 A between the catalytic site labeled with ISA and a GTP site labeled with 5'-FSBa epsilon A was measured from the quenching of salicylate donor fluorescence in the presence of the SBa epsilon A acceptor on a doubly labeled enzyme. The average distance between the ADP and GTP sites was previously measured as 18 A [Jacobson, M. A., & Colman, R. F. (1983) Biochemistry 22, 4247-4257], indicating that the regulatory sites of glutamate dehydrogenase are closer to each other than to the catalytic site.     

Reaction of the 2',3'-dialdehyde derivative of NADPH at a nucleotide site of bovine liver glutamate dehydrogenase

The 2',3'-dialdehyde derivative of NADPH (oNADPH) acts as a coenzyme for the reaction catalyzed by bovine liver glutamate dehydrogenase. Incubation of 250 microM oNADPH with enzyme for 300 min at 30 degrees C and pH 8.0 yields covalent incorporation of 1.0 mol of oNADPH/mol of enzyme subunit. The modified enzyme has a functional catalytic site and is activated by ADP, but is no longer inhibited by high NADH concentrations and exhibits decreased sensitivity to GTP inhibition. Using the change in inhibition by 600 microM NADH or 1 microM GTP to monitor the reaction leads to rate constants of 44.0 and 41.5 min-1 M-1, respectively, suggesting that loss of inhibition by the two regulatory compounds results from reaction by oNADPH at a single location. The oNADPH incorporation is proportional to the decreased inhibition by 600 microM NADH or 1 microM GTP, extrapolating to less than 1 mol of oNADPH/mol of subunit when the maximum change in NADH or GTP inhibition has occurred. Modified enzyme is still 93% inhibited at saturating levels of GTP, although its K1 is increased 20-fold to 4.6 microM. The kinetic effects caused by oNADPH are not prevented by alpha-ketoglutarate, ADP, 5 mM NADH, or 200 microM GTP alone, but are prevented by 5 mM NADH with 200 microM GTP. Incorporation of oNADPH into enzyme at 255 min is 0.94 mol/mol of peptide chain in the absence of ligands but only 0.53 mol/mol of peptide chain in the presence of the protectants 5 mM NADH plus 200 microM GTP. These results indicate that oNADPH modifies specifically about 0.4-0.5 sites/enzyme subunit or about 3 sites/enzyme hexamer and that reaction occurs at a GTP-dependent inhibitory NADH site of glutamate dehydrogenase.     

Guanosine 5'-O-[S-(3-bromo-2-oxopropyl)]thiophosphate: a new reactive purine nucleotide analog labeling Met-169 and Tyr-262 in bovine liver glutamate dehydrogenase.

A new guanosine nucleotide has been synthesized and characterized: guanosine 5'-O-[S-(3-bromo-2-oxopropyl)]thiophosphate (GMPSBOP), with a reactive functional group which can be placed at a position equivalent to the pyrophosphate region of GTP. This new analog is negatively charged at neutral pH and is similar in size to GTP. GMPSBOP has been shown to react with bovine liver glutamate dehydrogenase with an incorporation of 2 mol of reagent/mol of subunit. The modification reaction desensitizes the enzyme to inhibition by GTP, activation by ADP, and inhibition by high concentrations of NADH, but does not affect the catalytic activity of the enzyme. The rate constant for reaction of GMPSBOP with the enzyme exhibits a nonlinear dependence on reagent concentration with KD = 75 microM. The addition to the reaction mixture of alpha-ketoglutarate, GTP, ADP, or NADH alone results in little decrease in the rate constant, but the combined addition of 5 mM NADH with 0.4 mM GTP or with 10 mM alpha-ketoglutarate reduces the reaction rate approximately 6-fold. GMPSBOP modifies peptides containing Met-169 and Tyr-262, of which Tyr-262 is not critical for the decreased sensitivity of the enzyme toward allosteric ligands. The presence of 0.4 mM GTP plus 5 mM NADH protects the enzyme against reaction at both Met-169 and Tyr-262, but yields enzyme with 1 mol of reagent incorporated/mol of subunit which is modified at an alternate site, Met-469. In the presence of 0.2 mM GTP + 0.1 mM NADH, protection against modification of Tyr-262, but only partial protection against labeling of Met-169, is observed. In contrast, the presence of 10 mM alpha-ketoglutarate + 5 mM NADH protect only against reaction with Met-169. The results suggest that GMPSBOP reacts at the GTP-dependent NADH regulatory site [Lark, R. H., & Colman, R. F. (1986) J. Biol. Chem. 261, 10659-10666] of bovine liver glutamate dehydrogenase, which markedly affects the sensitivity of the enzyme to GTP inhibition. The reaction of GMPSBOP with Met-169 is primarily responsible for the altered allosteric properties of the enzyme.     

Adenosine 5'-0-[S-(4-succinimidyl-benzophenone)thiophosphate]: a new photoaffinity label of the allosteric ADP site of bovine liver glutamate dehydrogenase

By reaction of adenosine 5'-monothiophosphate with benzophenone-4-maleimide, we synthesized adenosine 5'-O-[S-(4-succinimidyl-benzophenone)thiophosphate] (AMPS-Succ-BP) as a photoreactive ADP analogue. Bovine liver glutamate dehydrogenase is known to be allosterically activated by ADP, but the ADP site has not been located in the crystal structure of the hexameric enzyme [Peterson, P. E., and Smith, T. J. (1999) Structure 7, 769-782]. In the dark, AMPS-Succ-BP reversibly activates GDH. Irradiation of the complex of glutamate dehydrogenase and AMPS-Succ-BP at lambda >300 nm causes a time-dependent, irreversible 2-fold activation of the enzyme. The k(obs) for photoactivation shows nonlinear dependence on the concentration of AMPS-Succ-BP, with K(R) = 4.9 microM and k(max) = 0.076 min(-)(1). The k(obs) for photoreaction by 20 microM AMPS-Succ-BP is decreased 10-fold by 200 microM ADP, but is reduced less than 2-fold by NAD, NADH, GTP, or alpha-ketoglutarate. Modified enzyme is no longer activated by ADP, but is still inhibited by GTP and high concentrations of NADH. These results indicate that reaction of AMPS-Succ-BP occurs within the ADP site. The enzyme incorporates up to 0.5 mol of [(3)H]AMPS-Succ-BP/mol of enzyme subunit or 3 mol of reagent/mol of hexamer. The peptide Lys(488)-Glu(495) has been identified as the only reaction target, and the data suggest that Arg(491) is the modified amino acid. Arg(491) (in the C-terminal helix close to the GTP #2 binding domain of GDH) is thus considered to be at or near the enzyme's allosteric ADP site. On the basis of these results, the AMPS-Succ-BP was positioned within the crystal structure of glutamate dehydrogenase, where it should also mark the ADP binding site of the enzyme.     

Affinity labeling of the reduced diphosphopyridine nucleotide inhibitory site of glutamate dehydrogenase by 6-[(4-bromo-2,3-dioxobutyl)thio]-6-deaminoadenosine 5'-diphosphate

Bovine liver glutamate dehydrogenase reacts covalently with the new adenosine analogue 6-[(4-bromo-2,3-dioxobutyl)thio]-6-deaminoadenosine 5'-diphosphate with incorporation of about 1 mol of reagent/mol of enzyme subunit. Modified enzyme completely loses its normal ability to be inhibited by high concentrations of reduced diphosphopyridine nucleotide (DPNH) (greater than 100 microM), which binds at a regulatory site distinct from the catalytic site; however, the modified enzyme retains its full activity when assayed at 100 microM DPNH in the absence of allosteric compounds. The enzyme is still activated by ADP, is inhibited by GTP (albeit at higher concentrations), and binds 1.5-2 mol of [14C]GTP/subunit. A plot of initial velocity vs. DPNH concentration for the modified enzyme, in contrast to the native enzyme, followed Michaelis-Menten kinetics. The rate constant (k) for loss of DPNH inhibition (as measured at 0.6 mM DPNH) exhibits a nonlinear dependence on reagent concentration, suggesting a reversible binding of reagent (Kd = 0.19 mM) prior to irreversible modification. At 0.1 mM 6-[(4-bromo-2,3-dioxobutyl)thio]-6-deaminoadenosine 5'-diphosphate, k = 0.036 min-1 and is not affected by alpha-ketoglutarate, 100 microM DPNH, or GTP alone but is decreased to 0.0094 min-1 by 5 mM DPNH and essentially to zero by 5 mM DPNH plus 100 microM GTP. Incorporation after incubation with 0.25 mM 6-[(4-bromo-2,3-dioxobutyl)thio]-6-deaminoadenosine 5'-diphosphate for 2 h at pH 7.1 is 1.14 mol/mol of subunit in the absence but only 0.24 mol/mol of subunit in the presence of DPNH plus GTP.(ABSTRACT TRUNCATED AT 250 WORDS)     

Affinity labeling of pig lung glutathione S-transferase pi by 4-(fluorosulfonyl)benzoic acid

The compound 4-(fluorosulfonyl)benzoic acid (4-FSB) functions as an affinity label of the dimeric pig lung pi class glutathione S-transferase yielding a completely inactive enzyme. Protection against inactivation is provided by glutathione-based ligands, suggesting that the reaction target is near or part of the glutathione binding site. Radioactive 4-FSB is incorporated to the extent of 1 mol per mole of enzyme subunit. Peptide mapping revealed that 4-FSB reacts with two tyrosine residues in the ratio 69% Tyr7 and 31% Tyr106. The ratio is not changed by the addition of ligands. The results suggest that only one of the tyrosine residues can be labeled in the active site of a given subunit; i.e., reactions with Tyr7 and Tyr106 are mutually exclusive. We propose that the difference in labeling of these tyrosine residues is related to their pKa values, with Tyr7 exhibiting the lower pKa. The modified enzyme no longer binds to a S-hexylglutathione-agarose affinity column, even when only one of the active sites contains 4-FSB; these results may reflect interaction between the subunits. We conclude that Tyr7 and Tyr106 of the pig lung class pi glutathione S-transferase are important for function and are located at or close to the substrate binding site of the enzyme.     

Affinity labeling of the inhibitory DPNH site of bovine liver glutamate dehydrogenase by 5'-fluorosulfonylbenzoyl adenosine.

A new adenosine analogue has been synthesized, 5'-fluorosulfonylbenzoyl adenosine, which reacts covalently with bovine liver glutamate dehydrogenase with the incorporation of approximately 1 mol of 5'-sulfonylbenzoyl adenosine per peptide chain. Native glutamate dehydrogenase is known to be inhibited by relatively high concentrations of DPNH by binding to a second noncatalytic site; the major change in the kinetic characteristics of the modified enzyme is a total loss of this inhibition by DPNH. The modified enzyme retains full catalytic activity as measured in the absence of allosteric ligands, is still inhibited more than 90% by GTP, and is activated normally by ADP. These results demonstrate that the catalytic as well as the GTP and ADP regulatory sites are distinct from the inhibitory DPNH site. The rate constant for reaction of 5'-fluorosulfonylbenzoyl adenosine is decreased by high concentrations of DPNH alone or by DPNH plus GTP, but not by the substrate alpha-ketoglutarate, the coenzymes DPN or TPNH, or the regulators ADP or GTP alone. These observations are consistent with the postulate that the 5'-fluorosulfonylbenzoyl adenosine attacks exclusively the second inhibitory DPNH site. The DPNH inhibition is abolished when an average of only 0.5 mol of 5'-sulfonylbenzoyl adenosine per peptide chain has been incorporated. The structure of 5'-fluorosulfonylbenzoyl adenosine is critical in determining the course of the modification reaction. The smaller compound p-fluorosulfonylbenzoic acid does not affect the kinetic characteristics of the enzyme, and the isomeric compound 3'-fluorosulfonylbenzoyl adenosine produces a different pattern of changes in the regulatory properties (Pal. P. K., Wechter, W. J., and Colman, R. F. (1975) Biochemistry 14, 707-715). Indeed, enzyme which has combined stoichiometrically with 5'-fluorosulfonylbenzoyl adenosine is still able to react with 3'-fluorosulfonylbenzoyl adenosine; thus, the two adenosine analogues appear to react at distinct sites on glutamate dehydrogenase. It is proposed that 5'-fluorosulfonylbenzoyl adenosine will be complementary to 3'-fluorosulfonylbenzoyl adenosine as a general affinity label for dehydrogenases as well as other classes of enzymes which use adenine nucleotides as substrates or regulators.     

Purification and properties of glutamate dehydrogenase from the mantle muscle of the squid, Loligo pealeii. Role of the enzyme in energy production from amino acids.

1. The activity of glutamate dehydrogenase was measured in the tissues of the squid, Loligo pealeii. The enzyme occurs in high activity in digestive pouch, systemic heart, and all muscle tissues. 2. Glutamate dehydrogenase from mantle muscle is located intra-mitochondrially, has a molecular weight of 310,000, and is electrophoretically similar to the enzyme from all other squid tissues. 3. The enzyme from mantle muscle was purified 40-fold by elution from DEAE-cellulose and used for kinetic studies. The enzyme is NAD+-specific, activated by ADP, AMP, and leucine, and inhibited by GTP, GDP, ATP, and reaction products (in particular NADH). 4. Squid glutamate dehydrogenase shows an almost absolute dependence on ADP. The purified enzyme is activated over 100-fold by saturating concentrations of ADP (Ka = 0,75 7M); The pH optima are also altered significantly by ADP. 5. The enzyme appears to be kinetically adapted to favour glutamate oxidation in comparison to glutamate dehydrogenase from other resources. The evidence indicates that the primary role of glutamate dehydrogenase in squid mantle muscle is in regulating the catabolism of amino acids for energy production.     

Inactivation of glutamate dehydrogenase and glutamate synthase from Bacillus megaterium by phenylglyoxal, butane-2,3-dione and pyridoxal 5''-phosphate.

Reaction of phenylglyoxal with glutamate dehydrogenase (EC 1.4.1.4), but not with glutamate synthase (EC 2.6.1.53), 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.     

Modification of glutamate dehydrogenase by pyridoxal-5'-phosphate. Study of the cooperative type of inhibition by GTP

The character of allosteric inhibition of glutamate dehydrogenase by GTP was studied. The derivative of the enzyme not capable of being polymerized was taken as a model. It was shown that: in the absence of NADH every protomer of this derivative can bind one molecule of GTP; in the presence of NADH the additional binding site for GTP was induced; the modification of the enzyme derivative by pyridoxal-5-phosphate in the presence of NADH and alpha-ketoglutarate blocked the NADH-induced GTP binding site and the disappearance of positive kinetic cooperativity induced by GTP was observed; to achieve the inhibitory action of GTP the binding of the effector to only one (NADH-induced) site was enough; the role of GTP binding to the NADH-induced site is to provide better affinity of the effector to the "inhibitory" centre; the positive kinetic cooperativity of inhibition of glutamate dehydrogenase by GTP depends probable on the cooperative character of interaction between the two molecules of GTP to each protomer of the enzyme.     

Affinity labeling of a glutamyl peptide in the coenzyme binding site of NADP+-specific glutamate dehydrogenase of Salmonella typhimurium by 2-[(4-bromo-2,3-dioxobutyl)thio]-1,N6-ethenoadenosine 2',5'-bisphosphate

NADP+-specific glutamate dehydrogenase from Salmonella typhimurium, cloned and expressed in Escherichia coli, has been purified to homogeneity. The nucleotide sequence of S. typhimurium gdhA was determined and the amino acid sequence derived. The nucleotide analogue 2-[(4-bromo-2,3-dioxobutyl)thio]-1,N6-ethenoadenosine 2',5'-bisphosphate (2-BDB-T epsilon A-2',5'-DP) reacts irreversibly with the enzyme to yield a partially inactive enzyme. After about 60% loss of activity, no further inactivation is observed. The rate of inactivation exhibits a nonlinear dependence on 2-BDB-T epsilon A-2',5'-DP concentration with kmax = 0.160 min-1 and KI = 300 microM. Reaction of 200 microM 2-BDB-T epsilon A-2',5'-DP with glutamate dehydrogenase for 120 min results in the incorporation of 0.94 mol of reagent/mol of enzyme subunit. The coenzymes, NADPH and NADP+, completely protect the enzyme against inactivation by the reagent and decrease the reagent incorporation from 0.94 to 0.5 mol of reagent/mol enzyme subunit, while the substrate alpha-ketoglutarate offers only partial protection. These results indicate that 2-BDB-T epsilon A-2',5'-DP functions as an affinity label of the coenzyme binding site and that specific reaction occurs at only about 0.5 sites/enzyme subunit or 3 sites/hexamer. Glutamate dehydrogenase modified with 200 microM 2-BDB-T epsilon A-2',5'-DP in the absence and presence of coenzyme was reduced with NaB3H4, carboxymethylated, and digested with trypsin. Labeled peptides were purified by high performance liquid chromatography and characterized by gas phase sequencing. Two peptides modified by the reagent were isolated and identified as follows: Phe-Cys(CM)-Gln-Ala-Leu-Met-Thr-Glu-Leu-Tyr-Arg and Leu-Cys(CM)-Glu-Ile-Lys. These two peptides were located within the derived amino acid sequence as residues 146-156 and 282-286. In the presence of NADPH, which completely prevents inactivation, only peptide 146-156 was labeled. This result indicates that modification of the pentapeptide causes loss of activity. Glutamate 284 in this peptide is the probable reaction target and is located within the coenzyme binding site.     

The effect of modifying lysine-126 on the physical, catalytic and regulatory properties of bovine liver glutamate dehydrogenase

1. The reaction of 4-iodoacetamidosalicylate with bovine liver glutamate dehydrogenase is dependent on pH. The pH-activity curve is bell-shaped and can be described by apparent pK values of 7.8+/-0.2 and 9.1+/-0.2. 2. Enzyme in which lysine-126 has been modified by 4-iodoacetamidosalicylate has unaltered sedimentation characteristics except when measured in the presence of GTP and NADH. 3. GTP binding to the inhibited enzyme is unaltered. However, GTP can no longer promote the binding of a second molecule of NADH, since this is already bound to the inhibited enzyme without GTP. 4. The equilibrium binding of ADP, GTP, NAD-sulphite and NADH (when measured at low concentrations) was largely unchanged by modification. 5. The number of binding sites for 2-oxoglutarate to the enzyme-NADH complex were decreased by 60% in an enzyme that has been inhibited by 70%.     

Identification of an essential lysine in porcine heart mitochondrial malate dehydrogenase.

The reversible inactivation of porcine heart mitochondrial malate dehydrogenase by pyridoxal 5'-phosphate yields an irreversible modification upon sodium borohydride reduction. A 200-fold molar excess of pyridoxal-5'-P over enzyme results in inactivation to the extent of 54%, and incorporation of 5.7 mol of inactivator per mol of enzyme. The same inactivation carried out in the presence of 80 mM coenzyme, NADH, produces malate dehydrogenase which is approximately 94% active and contains 4.6 mol of pyridoxal-5'-P per mol of enzyme. The incorporation difference between inactivated and protected samples suggests, for total inactivation, the modification of 2 residues per mol of enzyme (i.e. 1 residue per subunit, or 1 per enzymatic active site). This specificity was confirmed by the isolation of a single pyridoxyl-5'-P-labeled "difference peptide" obtained by comparison of the Dowex 1-X2 elution profiles of tryptic digests of protected and inactivated samples, respectively. Amino acid analysis of the peptide demonstrated the presence of N6-pyridoxyl-L-lysine (Lys(Pyx)), establishing the existence of an essential lysing residue in the active center of malate dehydrogenase. The amino acid sequence of the active center hexapeptide has been determined to be: H2NLys(Pyx)Pro-Gly-Met-Thr-Arg-COOH.     

A chromophoric and fluorescent analog of GTP, 2',3'-O-(2,4,6-trinitrocyclohexadienylidene)-GTP, as a spectroscopic probe for the GTP inhibitory site of liver glutamate dehydrogenase

The ribose-modified chromophoric and fluorescent analog of ATP, 2',3'-O-(2,4,6-trinitrocyclohexadienylidene)-ATP (TNP-ATP) (Hiratsuka, T., and Uchida, K. (1973) Biochim. Biophys. Acta 320, 635-647 and Hiratsuka, T. (1976) Biochim. Biophys. Acta 453, 293-297) has been widely used as an ATP analog for various ATPases. Although the corresponding analog of GTP,2',3'-O-(2,4,6-trinitrocyclohexadienylidene)-GTP (TNP-GTP) should be useful for the study of various GTP-requiring enzymes, it is difficult to prepare TNP-GTP by the conventional method. In the present study, we succeeded in the synthesis of TNP-GTP with the use of an alternative method. The analogs of GDP, GMP, and guanyl-5'-yl imidodiphosphate (Gpp(NH)p) were also synthesized. Visible absorption and fluorescent properties of TNP-GTP, TNP-GDP, TNP-GMP, and TNP-Gpp(NH)p were quite similar to those of TNP-ATP. TNP-GTP was found to be able to replace GTP as an inhibitor for bovine liver glutamate dehydrogenase. The enzyme was inhibited by TNP-GTP to a maximum extent of 54% at saturating concentrations of the analog with a KI of 2.7 microM. TNP-Gpp(NH)p and other ribose-modified fluorescent analogs of GTP,3'-O-anthraniloyl-GTP and 3'-O-(N-methylanthraniloyl)-GTP (Hiratsuka, T. (1983) Biochim. Biophys. Acta 742, 496-508), also inhibited the enzymatic activity. Binding of TNP-GTP to the enzyme was characterized by a 5.6-fold enhancement in analog fluorescence. In the presence of NADH, the limiting fluorescence enhancement of the bound analog decreased to 2.7-fold. As determined by fluorometric titration, the maximum number of TNP-GTP binding sites on the enzyme was 1.9 mol/mol of subunit with a KD of 0.66 microM in the absence of NADH and 2.2 mol/mol of subunit with two KD values of 0.11 and 0.71 microM in the presence of NADH. These observations suggest that NADH binding increases the affinity of only 1 mol of the 2 mol of TNP-GTP bound to the enzyme. These spectroscopic and biological properties of TNP-GTP should make this analog useful as a chromophoric and fluorescent probe for studies not only of glutamate dehydrogenase but also of various GTP-requiring enzymes, which have a high specificity for the base moiety of GTP.     

Affinity labeling of bovine adrenal 3 beta-hydroxysteroid dehydrogenase/steroid isomerase by 5'-[p-(fluorosulfonyl)benzoyl]adenosine

Incubation of bovine adrenal 3 beta-hydroxysteroid dehydrogenase/steroid isomerase with 5'-[p-(fluorosulfonyl)benzoyl]adenosine (5'-FSBA) results in the inactivation of the 3 beta-hydroxysteroid dehydrogenase enzyme activity following pseudo-first-order kinetics. A double-reciprocal plot of 1/kobs versus 1/[5'-FSBA] yields a straight line with a positive y intercept, indicative of reversible binding of the inhibitor prior to an irreversible inactivation reaction. The dissociation constant (Kd) for the initial reversible enzyme-inhibitor complex is estimated at 0.533 mM, with k2 = 0.22 min-1. The irreversible inactivation could be prevented by the presence of NAD+ during the incubation, indicating that 5'-FSBA inactivates the 3 beta-hydroxysteroid dehydrogenase activity by reacting at the NAD+ binding site. Although the enzyme was inactivated by incubation with 5'-FSBA, no incorporation of the inhibitor was found in labeling studies using 5'-[p-(fluorosulfonyl)benzoyl] [14C]adenosine. However, the inactivation of 3 beta-hydroxysteroid dehydrogenase activity caused by incubation with 5'-FSBA could be completely reversed by the addition of dithiothreitol. This indicates the presence of at least two cysteine residues at or in the vicinity of the NAD+ binding site, which may form a disulfide bond catalyzed by the presence of 5'-FSBA. The intramolecular cysteine disulfide bridge was found between the cysteine residues in the peptides 274EWGFCLDSR282 and 18IICLLVEEK26, by comparing the [14C]iodoacetic acid labeling before and after recovering the enzyme activity upon the addition of dithiothreitol.     

Modification of glutamate dehydrogenase by pyridoxal-5'-phosphate. Study of the structural organisation of the hexamer and its possible role in the realization of GTP action

The catalytic and regulator properties of glutamate dehydrogenase by modification of Lys-126 residue by puridoxal-5'-phosphate was studied. The phosphopyridoxyl derivative of the enzyme with blocked NADH-induced binding site of GTP not capable of being polymerized was taken as a model. It was shown that: blocking the epsilon-amino group of Lys-126 residue brings to a simultaneous inactivation of the enzyme and desensibilization of its residual activity to GTP action; the modification of Lys-126 residue and resulting inactivation of the enzyme and desensibilization to GTP action were non-cooperative processes, with equal values of pseudofirst order rate constants; modification of Lys-126 residue of any of hexamer's protomer results in the desensibilization to GTP action on one of the contacting, catalytically active protomers. The experimental dependence of the inhibition degree of the enzyme by GTP upon the average number of modified residues of Lys-126 is explained by the model of the hexamer of glutamate dehydrogenase with identical interlocation of any of the protomers in relation to the one in contact.     

The importance of arginine residues in the catalytic and regulatory functions of bovine-liver glutamate dehydrogenase.

Bovine liver glutamate dehydrogenase reacts rapidly with 2,3-butanedione to yield modified enzyme with 29% of its original maximum activity, but no change in its Michaelis constants for substrates and coenzymes. No significant reduction in the inactivation rate is produced by the addition of the allosteric activator ADP or inhibitor GTP, while partial protection against inactivation is provided by the coenzyme NAD+ or substrate 2-oxoglutarate when added separately. The most marked decrease in the rate of inactivation (about 10-fold) is provided by the combined addition of NAD+ and 2-oxoglutarate, suggesting that modification takes place in the region of the active site. Reaction with 2,3-butanedione also results in loss of the ability of the enzyme to be activated by ADP. Addition of ADP (but not NAD+, 2-oxoglutarate or GTP) to the incubation mixture protects markedly against the loss of activatability of ADP. It is concluded that 2,3-butanedione produces two distinguishable effects on glutamate dehydrogenase: a relatively specific modification of the regulatory ADP site and a distinct modification in the active center. Reaction of two arginyl residues per peptide chain appears to be responsible for disruption of the ADP activation property of the enzyme, while alteration of a maximum of five arginyl residues can be related to the reduction of maximum catalytic activity. Electrostatic interactions between the positively charged arginine groups and the negatively charged substrate, coenzyme and allosteric purine nucleotide may be important for the normal function of glutamate dehydrogenase.