Affinity labeling of a regulatory site of bovine liver glutamate dehydrogenase.

A new adenosine analog, 3'-p-fluorosulfonyl-benzoyladenosine (3'-FSBA), has been synthesized which reacts covalently with bovine liver glutamate dehydrogenase. Native glutamate dehydrogenase is activated by ADP and inhibited by high concentrations of DPNH. Both of these effects are irreversibly decreased upon incubation of the enzyme with the adenosine analog, 3'-p-fluorosulfonyl-benzoyladenosine (3'-FSBA), while the intrinsic enzymatic activity as measured in the absence of regulatory compounds remains unaltered. A plot of the rate constant for modification as a function of the 3'-FSBA concentration is not linear, suggesting that the adenosine derivative binds to the enzyme (Ki equals 1.0 times 10-4 M) prior to the irreversible modification. Protection against modification by 3'-FSBA is provided by ADP and by high concentrations of DPNH, but not by the inhibitor GTP, the substrate alpha-keto glutarate, the coenzyme TPNH, or low concentrations of the coenzyme DPNH. The isolated altered enzyme contains approximately 1 mol of sulfonylbenzoyladenosine per peptide chain, indicating that a single specific regulatory site has reacted with 3'-tfsba. the modified enzyme exhibits normal Michaelis constants for its substrates and is still inhibited by GTP, albeit at a higher concentration, but it is not inhibited by high concentrations of DPNH. Although ADP does not appreciably activate the modified enzyme, it does (as in the case of the native enzyme) overcome the inhibition of the modified enzyme by GTP. These results suggest that ADP can bind to the modified enzyme, but that its ability to activate is affected indirectly by the modification of the adjacent tdpnh inhibitory site. It is proposed that the regulatory sites for ADP and DPNH are partially overlapping and that 3'FSBA functions as a specific affinity label for the DPNH inhibitory site of glutamate dehydrogenase. It is anticipated that 3'-p-fluorosulfonylbenzolyadenosine may act as an affinity label of other dehydrogenases as well as of other classes of enzymes which use adenine nucleotides as substrates or regulators.     

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.     

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.     

Identification of the GTP binding site of human glutamate dehydrogenase by cassette mutagenesis and photoaffinity labeling

It has been reported that the hyperinsulinism-hyperammonemia syndrome is caused by mutations in glutamate dehydrogenase (GDH) gene that affects enzyme sensitivity to GTP-induced inhibition. To identify the GTP binding site(s) within human GDH, mutant GDHs at Tyr-266 or Lys-450 position were constructed by cassette mutagenesis. More than 90% of the initial activities were remained at the concentration of GTP up to 300 microm for the Lys-450 mutant GDHs regardless of their size, hydrophobicity, and ionization of the side chains, whereas the wild type GDH and the Tyr-266 mutant GDHs were completely inhibited by 30 microm GTP. The binding of GTP to the wild type GDH or the mutant GDHs was further examined by photoaffinity labeling with 8-[gamma-(32)P]azidoguanosine 5'-triphosphate (8-N(3)-GTP). Saturation of photoinsertion with 8-N(3)-GTP occurred apparent K(d) values near 20 microm for the wild type GDH or the Tyr-266 mutant GDH, and the photoinsertion of 8-N(3)-[gamma-(32)P]GTP was significantly decreased in the presence of 300 microm GTP. Unlike the wild type GDH or the Tyr-266 mutant GDH, less than 10% of photoinsertion was detected in the Lys-450 mutant GDH, and the photoinsertion was not affected by the presence of 300 microm GTP. The results with cassette mutagenesis and photoaffinity labeling demonstrate selectivity of the photoprobe for the GTP binding site and suggest that Lys-450, but not Tyr-266, is required for efficient binding of GTP to GDH. Interestingly, studies of the steady-state velocity showed that both the wild type GDH and the Tyr-266 mutant GDHs were inhibited by ATP at concentrations between 10 and 100 microm, whereas less than 10% of the initial activities of the Lys-450 mutant GDHs were diminished by ATP. These results indicate that Lys-450, but not Tyr-266, may be also responsible for the ATP inhibition; therefore, ATP bound to the GTP site.     

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 the allosteric site of fructose 1,6-bisphosphatase with an AMP analog

D-Fructose 1,6-bisphosphatase [EC 3.1.3.11, FBPase] is one of the key enzymes in glyconeogenesis and its activity is controlled by various effectors such as substrate, AMP and ATP. To analyze this complex regulation system, we tried an affinity labeling of FBPase with an AMP derivative, since AMP is a potent allosteric inhibitor of this enzyme. The results obtained are as follows. 1. To determine the functional groups which are essential for AMP as an inhibitor, inhibitory activities of some AMP derivatives were examined. These derivatives modified at the purine ring or phosphate group lost the activity while one modified at the ribose ring retained the ability to inhibit FBPase. This shows that an affinity labeling reagent should be an AMP derivative in which the ribose ring is modified. 2. 2',3'-Dialdehyde AMP (dial-AMP) was prepared by periodate oxidation of AMP and was reacted with FBPase. Under appropriate conditions, 1 mol of the reagent was incorporated per mol of enzyme subunit with a concomitant loss of enzyme activity. The reaction was prevented by the presence of AMP but not of ATP. The heat-stability, the kinetic parameters and the UV-absorption spectrum of the modified enzyme were all the same as those of native FBPase in the presence of AMP. Thus it was concluded that the allosteric AMP site in FBPase was modified specifically.     

Affinity labeling of the allosteric ADP activation site of NAD-dependent isocitrate dehydrogenase by 6-(4-bromo-2,3-dioxobutyl)thioadenosine 5'-diphosphate

Pig heart NAD-dependent isocitrate dehydrogenase is allosterically activated by ADP which reduces the Km of isocitrate. The new ADP analogue 6-(4-bromo-2,3-dioxobutyl)thioadenosine 5'-diphosphate (BDB-TADP) reacts irreversibly with the enzyme at pH 6.1 and 25 degrees C, causing a rapid loss of the ability of ADP to increase the initial velocity of assays conducted at low isocitrate concentrations and a slower inactivation measured using saturating isocitrate concentrations. The rate constant for loss of ADP activation exhibits a nonlinear dependence on BDB-TADP concentration; in the presence of 0.2 mM MnSO4, KI for the reversible enzyme-reagent complex is 0.069 mM with kmax at saturating reagent concentrations equal to 0.031 min-1. For reaction at the site causing overall inactivation, KI for the initial reversible enzyme-reagent complex is estimated to be 0.018 mM with kmax = 0.0083 min-1 in the presence of 0.2 mM MnSO4. Total protection against both reactions is provided by 1 mM ADP plus 0.2 mM MnSO4 or by 0.1 mM ADP plus 0.2 mM MnSO4 plus 0.2 mM isocitrate, but not by NAD, ATP, or ADP plus EDTA. The BDB-TADP thus appears to modify two distinct metal-dependent ADP-binding sites. Incubation of isocitrate dehydrogenase with 0.14 mM BDB-[beta-32P]TADP at pH 6.1 in the presence of 0.2 mM MnSO4 results in incorporation of 0.81 mol of reagent/mol of average subunit when the ADP activation is completely lost and the enzyme is 68% inactivated. The time-dependent incorporation is consistent with the postulate that covalent reaction of 0.5 mol of BDB-TADP/mol of average enzyme subunit causes complete loss of ADP activation, while reaction with another 0.5 mol of BDB-TADP would lead to total inactivation. The enzyme is composed of three distinct subunits in the approximate ratio 2 alpha:1 beta:1 gamma. The distribution of BDB-[beta-32P]TADP incorporated into modified enzyme is 63:30:7% for alpha:beta:gamma throughout the course of the reaction. These results indicate the 6-(4-bromo-2,3-dioxobutyl)thioadenosine 5'-diphosphate functions as an affinity label of two types of potential metal-dependent ADP sites of NAD-dependent isocitrate dehydrogenase and that these allosteric sites are present on two (alpha and beta) of the enzyme's three types of subunits.     

Affinity labelling of the allosteric site of the L-lactate dehydrogenase of Lactobacillus casei

We have recently described the addition of 2-keto-3-butynoic acid to flavin-free flavocytochrome b2, a reaction which leads to the loss of flavin-binding capacity ('inactivation') [D. Pompon and F. Lederer (1982) Eur. J. Biochem. 129, 143-137]. For total inactivation, the extrapolated incorporation value was 0.9 mol reagent/mol subunit. In this work we report the results of sequence studies which elucidate the nature of the modification. The modified protein was cleaved with cyanogen bromide and the peptides separated on Sephadex G-100 and SP-Sephadex C-25. 14C-labeled peptides were digested with trypsin and chymotrypsin and smaller labeled fragments purified by chromatography on Sephadex G-50 and thin-layer fingerprinting. It is shown that three cysteine residues are fractionally labeled with nearly complete mutual exclusion. Furthermore, a fraction of the modified peptides is found under the form of cross-linked fragments, where two cysteines have added to the same ketobutynoate molecule. Only two of the possible cross-links were found. These results show that the three cysteines are close to one another in space in the flavin-free enzyme and hence probably also in the holoenzyme. These results, combined with those obtained in the affinity labeling reaction of holoenzyme with bromopyruvate [Alliel et al. (1982) Eur. J. Biochem. 122, 553-558], show that the three residues are located in or close to the active site. Their possible role is discussed.     

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.     

Heat denaturation of bovine liver glutamate dehydrogenase.

Heat denaturation of bovine liver glutamate dehydrogenase occurred at 47 degrees with loss of enzyme activity and formation of inactive, insoluble protein. Fractional loss of catalytic activity coincided with alteration in protein fluorescence and solubility for a corresponding percentage of protein molecules. Operationally, at 50% denaturation, one-half of the total population of enzyme molecules is fully active catalytically and soluble and the other half of the protein molecule population is completely inactive catalytically and insoluble.     

Nickel-induced substrate inhibition of bovine liver glutamate dehydrogenase

The effects of nickel ions on reductive amination and oxidative deamination activities of bovine liver glutamate dehydrogenase (GDH) were examined kinetically by UV spectroscopy, at 27 degrees C, using 50 mM Tris, pH 7.8, containing 0.1 M NaCl. Kinetic analysis of the data obtained by varying NADH concentration indicated strong inhibition, presumably due to binding of the coenzyme to the regulatory site. In contrast, almost no inhibition was observed in the forward reaction. The fact that nickel ions have the capacity to enhance binding of NADH to the enzyme was confirmed by an electrochemical method using a modified glassy carbon electrode. Use of NADPH instead of NADH showed only a weak substrate inhibition, presumably related to lower affinity of NADPH for binding to the regulatory site. Lineweaver-Burk plots with respect to alpha-ketoglutarate and ammonium ions indicated substrate and competitive inhibition patterns in the presence of nickel ions, respectively. ADP at 0.2 mM concentration protected inhibition caused by nickel. These observations are explained in terms of formation of a nickel-NADH complex with a higher affinity for binding to the regulatory site in GDH, as compared with the situation where nickel is not present. Such effects may be important for regulation of GDH and other NADH-utilizing enzymes.     

Affinity labeling of bovine opsin by trans-retinoyl chloromethane

All-trans-retinoyl chloromethane is a potent irreversible inactivator of bovine opsin in retinal rod outer segments. The inactivation appears to be due to specific modification of the apoprotein at the 11-cis-retinaldehyde binding site. The reaction follows pseudo first order kinetics at 37 degrees C. A moderate dissociation constant for the initial reversible complex of 6.1 mM could be derived with a first order rate constant of 1.8x10(-1) min-1 for the conversion to the irreversible inactivated form. Native rhodopsin or rhodopsin regenerated from opsin by addition of 11-cis-retinaldehyde is completely protected against inactivation by trans-retinoyl chloromethane. All-trans-retinaldehyde does not provide this protection for the irreversible inactivation.     

Affinity labeling of the GDP/GTP binding site in Thermus thermophilus elongation factor Tu

Elongation factor Tu from Thermus thermophilus was treated successively with periodate-oxidized GDP or GTP and cyanoborohydride. Covalently modified cyanogen bromide or trypsin fragments of the protein were isolated, and the position of their modification was determined. Lysine residues 52 and 137 were heavily labeled, lysine-137 being considerably more reactive in the GTP form as compared to the GDP form of the protein. These residues are in the proximity of the GDP/GTP binding site. Lys-325 was also labeled, but to a lower extent. The part of the EF-Tu containing residue 52 is missing in crystallized EF-Tu.GDP from Escherichia coli [Jurnak, F. (1985) Science (Washington, D.C.) 230, 32-36]. These results place the part of T. thermophilus EF-Tu corresponding to the missing fragment in E. coli EF-Tu in the vicinity of the nucleotide binding site and allow its role in the interaction with aminoacyl-tRNA and elongation factor Ts to be evaluated. Cross-linking of EF-Tu.GDP by irradiation at 257 nm showed that a sequence of 10 amino acids residues which is found in the Thermus thermophilus elongation factor Tu but not in other homologous bacterial proteins is located in the vicinity of the GDP/GTP binding site.     

Kinetics of pressure-induced inactivation of bovine liver glutamate dehydrogenase

Kinetics of pressure-induced denaturation of bovine liver glutamate dehydrogenase (EC 1.4.1.3) were investigated in the pressure range 1.8-2.8 kbar by observing the residual activity after the pressure-release and the scattered light intensity during the incubation at high pressure. The residual activity decreased exponentially with the incubation time, whereas the scattered light intensity showed a bimodal profile indicating parallel aggregation and dissociation reactions. The latter suggested that two kinds of aggregates were formed during the incubation under pressure. The observed first-order rate constant for the inactivation, k obs, showed a minimum around 30 degrees C. These experimental results were interpreted in terms of the following reaction scheme; (formula; see text) where N represents the enzyme entity with native structure, D1 the partially denatured intermediate, D2 the irreversibly denatured state, and A1 and A2 the two kinds of aggregates, one of which (A1) is reversibly formed at an early stage of the incubation under high pressure. The apparent activation volume for the inactivation reaction was estimated to be delta V*app = -113 +/- 5 cm3 X mol-1 from the pressure dependence of k obs. The effect of coenzyme, NAD+, on the pressure-induced inactivation was also studied. The inactivation was retarded by the presence of the coenzyme, whereas the apparent activation volume for the holoenzyme (delta V*app = -104 +/- 2 cm3 X mol-1) did not differ significantly from that for the apoenzyme.     

Affinity labeling of the allosteric activator site(s) of spinach leaf ADP-glucose pyrophosphorylase

Pyridoxal-P has been shown to be an activator of the spinach leaf ADP-glucose pyrophosphorylase. It has a higher apparent affinity than the physiological activator 3-phosphoglycerate but only activates the enzyme activity 6-fold whereas 3-phosphoglycerate gives a 25-fold activation. Reductive phosphopyridoxylation of the spinach leaf enzyme results in enzyme having less dependence on the presence of activator for activity. Labeled pyridoxal-P is incorporated into both the 54- and 51-kilodalton subunits of the spinach leaf enzyme. The incorporation is inhibited by the presence of either 3-phosphoglycerate or the allosteric inhibitor, inorganic phosphate, thus suggesting that pyridoxal phosphate is covalently bound to the allosteric activator site. The pyridoxal phosphate is bound to an epsilon-amino group of a lysine residue. The phosphopyridoxylated enzyme is more resistant to phosphate inhibition than the unmodified form. The modified 51-kDa subunit has been digested with trypsin, and the peptide containing the labeled pyridoxal phosphate has been purified via high performance liquid chromatography and sequenced. Comparison of this sequence with the deduced amino acid sequence of a rice endosperm cDNA clone indicates that the putative allosteric site of the 51-kDa subunit is close to the carboxyl-terminal. This is in contrast to what had been demonstrated for the position of the activator site of the Escherichia coli ADP-glucose pyrophosphorylase which was shown to be close to the amino-terminal of the subunit.     

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)