Identification of amino acids modified by the bifunctional affinity label 5'-(p-(fluorosulfonyl)benzoyl)-8-azidoadenosine in the reduced coenzyme regulatory site of bovine liver glutamate dehydrogenase.

Bovine liver glutamate dehydrogenase reacts with the bifunctional affinity label 5'-(p-(fluorosulfonyl)benzoyl)-8-azidoadenosine (5'-FSBAzA) in a two-step process: a dark reaction yielding about 0.5 mol of -SBAzA/mol of subunit by reaction through the fluorosulfonyl moiety, followed by photoactivation of the azido group whereby covalently bound -SBAzA becomes cross-linked to the enzyme [Dombrowski, K. E., & Colman, R. F. (1989) Arch. Biochem. Biophys. 275, 302-308]. We now report that the rate constant for the dark reaction is not reduced by ADP or GTP, but it is decreased 7-fold by 2 mM NADH and 40-fold by 2 mM NADH + 0.2 mM GTP, suggesting that 5'-FSBAzA reacts at the GTP-dependent NADH inhibitory site. The amino acid residues modified in each phase of the reaction have been identified. Modified enzyme was isolated after each reaction phase, carboxymethylated, and digested with trypsin, chymotrypsin, or thermolysin. The digests were fractionated by chromatography on a phenylboronate agarose column followed by HPLC. Gas-phase sequencing of the labeled peptides identified Tyr190 as the major amino acid which reacts with the fluorosulfonyl group; Lys143 was also modified but to a lesser extent. The predominant cross-link formed during photolysis is between modified Tyr190 and the peptide Leu475-Asp476-Leu477-Arg478, which is located near the C-terminus of the enzyme. Thus, 5'-FSBAzA is effective in identifying critical residues distant in the linear sequence, but close within the regulatory nucleotide site of glutamate dehydrogenase.     

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.     

Covalent modification of both cAMP binding sites in cAMP-dependent protein kinase I by 8-azidoadenosine 3',5'-monophosphate

Reconstituted porcine cAMP-dependent protein kinase type I was labeled with 8-azidoadenosine 3',5'-monophosphate (8-N3cAMP) to study cyclic nucleotide binding and to identify amino acid residues that are either in or in close proximity to the cAMP binding sites. The photoaffinity analogue 8-N3cAMP behaved as cAMP itself with respect to cyclic nucleotide binding. For both cAMP and 8-N3cAMP, 2 mol of nucleotide was bound per mole of type I regulatory subunit monomer (RI), the apparent Kd's observed were approximately 10-17 nM on the basis of either Millipore filtration assays, equilibrium dialysis, or ammonium sulfate precipitation, Scatchard plots showed positive cooperativity, and (4) the Hill coefficients were approximately 1.5-1.6. After photolysis and addition of an excess of cAMP, approximately 1 mol of 8-N3cAMP/mol of RI monomer was covalently incorporated. Tryptic digestion of the labeled protein revealed that two unique tryptic peptides were modified. Proline-271 and tyrosine-371 were identified as the two residues that were covalently modified by 8-N3cAMP in RI. These results contrast with the type II regulatory subunit (RII) where 8-N3cAMP modified covalently a single tyrosine residue [Kerlavage, A. R., & Taylor, S. S. (1980) J. Biol. Chem. 255, 8483-8488]. RI contains two adjacent regions of sequence homology in the COOH-terminal fragment that binds two molecules of cAMP.(ABSTRACT TRUNCATED AT 250 WORDS)     

Isolation of the glutamyl peptide labeled by the nucleotide analogue 2-(4-bromo-2,3-dioxobutylthio)-1,N(6)-ethenoadenosine 2',5'-biphosphate in the active site of NADP+-specific isocitrate dehydrogenase

2-(4-Bromo-2,3-dioxobutylthio)-1,N(6)-ethenoadenosine 2',5'-bisphosphate (2-BDB-T epsilon A-2',5'-DP) is an affinity label for the coenzyme-binding site of pig heart NADP+-dependent isocitrate dehydrogenase. Specific reaction occurs at the coenzyme site with an incorporation of 0.5 mol of reagent/mol of enzyme subunit (i.e. modification of only one subunit of the dimeric enzyme) (Bailey, J.M., and Colman, R.F. (1985) Biochemistry 24, 5367-5377). Modified enzyme, prepared by incubating 1 mg/ml isocitrate dehydrogenase with 75 microM 2-BDB-T epsilon A-2',5'-DP in the absence and presence of substrate or coenzyme, was reduced with NaBH4, carboxymethylated, and digested with trypsin. Nucleotidyl peptides were isolated by chromatography on DEAE-cellulose, followed by treatment with acid phosphatase (to decrease the negative charge by removing the phosphate groups from covalently bound reagent) and rechromatography on the same DEAE-cellulose column. The isolated peptides were characterized by amino acid analysis, dansylation, and gas-phase sequencing. A single triskaidekapeptide corresponding to modification of the coenzyme site by 2-BDB-T epsilon A-2',5'-DP was identified as: Asp-Leu-Ala-Gly-X-Ile-His-Gly-Leu-Ser-Asn-Val-Lys. Additional evidence indicated that X is a glutamate residue derivatized by 2-BDB-T epsilon A-2',5'-DP.     

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.     

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.     

Competitive inhibition of glutamate dehydrogenase reaction

Irrespective of their pyridine nucleotide specificity, all glutamate dehydrogenases share a common chemical mechanism that involves an enzyme bound 'iminoglutarate' intermediate. Three compounds, structurally related to this intermediate, were tested for the inhibition of purified NADP-glutamate dehydrogenases from two Aspergilli, as also the bovine liver NAD(P)-glutamate dehydrogenase. 2-Methyleneglutarate, closely resembling iminoglutarate, was a potent competitive inhibitor of the glutamate dehydrogenase reaction. This is the first report of a non-aromatic structure with a better glutamate dehydrogenase inhibitory potency than aryl carboxylic acids such as isophthalate. A suitably located 2-methylene group to mimic the iminium ion could be exploited to design inhibitors of other amino acid dehydrogenases.     

N-arylazido-beta-alanyl-NAD+, a new NAD+ photoaffinity analogue. Synthesis and labeling of mitochondrial NADH dehydrogenase

N-Arylazido-beta-alanyl-NAD+ [N3'-O-(3-[N-(4-azido-2-nitrophenyl)amino]propionyl)NAD+] has been prepared by alkaline phosphatase treatment of arylazido-beta-alanyl-NADP+ [N3'-O-(3-[N-(4-azido-2-nitrophenyl)amino]propionyl)NADP+]. This NAD+ analogue was found to be a potent competitive inhibitor (Ki = 1.45 microM) with respect to NADH for the purified bovine heart mitochondrial NADH dehydrogenase (EC 1.6.99.3). The enzyme was irreversibly inhibited as well as covalently labeled by this analogue upon photoirradiation. A stoichiometry of 1.15 mol of N-arylazido-beta-alanyl-NAD+ bound/mol of enzyme, at 100% inactivation, was determined from incorporation studies using tritium-labeled analogue. Among the three subunits, 0.85 mol of the analogue was bound to the Mr = 51,000 subunit, and each of the two smaller subunits contained 0.15 mol of the analogue when the dehydrogenase was completely inhibited upon photolysis. Both the irreversible inactivation and the covalent incorporation could be prevented by the presence of NADH during photolysis. These results indicate that N-arylazido-beta-alanyl-NAD+ is an active-site-directed photoaffinity label for the mitochondrial NADH dehydrogenase, and are further evidence that the Mr = 51,000 subunit contains the NADH binding site. Previous studies using A-arylazido-beta-alanyl-NAD+ [A3'-O-(3-[N-(4-azido-2-nitrophenyl)amino]propionyl)NAD+] demonstrated that the NADH binding site is on the Mr = 51,000 subunit [Chen, S., & Guillory, R. J. (1981) J. Biol. Chem. 256, 8318-8323]. Results are also presented to show that N-arylazido-beta-alanyl-NAD+ binds the dehydrogenase in a more effective manner than A-arylazido-beta-alanyl-NAD+.     

The reaction of bovine glutamate dehydrogenase with periodate-oxidised ADP

1. The reactive analogue oADP produced by periodate oxidation of ADP has been studied as a potential affinity label for the enzyme bovine glutamate dehydrogenase, using circular dichroism (CD) difference spectroscopy to monitor specific binding. 2. The analogue binds stoichiometrically, rapidly and reversibly to the adenine nucleotide binding site with Kd approximately equal to 12 microM (20 degrees C, pH 7) with characteristic intensification of the adenine nucleotide CD at 260 nm. 3. This complex is unstable and decays with a half-life of about 1.5 h; the analogue then becomes attached as a Schiff base to a number of subsidiary sites, including the enzyme active site, with partial inactivation of the enzyme. 4. Depending upon initial concentration of oADP, the enzyme activity is progressively lost during the slow reaction; following borohydride reduction, up to four molecules of analogue are bound/subunit. 5. Protection against loss of enzyme activity is afforded by the coenzyme NAD+ plus glutarate or L-hydroxyglutarate (an effective inhibitor), or by glutarate alone, but not by NAD+ alone. 6. Spectroscopic and protection studies indicate that after the decay of the specific CD signal, the enzyme retains the capacity to bind ADP, but that this is progressively lost in parallel with decay of enzymic activity. 7. The results are consistent with proximity or functional interaction between the adenine nucleotide site and the coenzyme binding portion of the active site. 8. Thus oADP does not act as a true affinity label for the adenine nucleotide binding site, but the reaction subsequent to binding at that site shows some specificity directed towards the active site.     

Determination of the free-energy coupling between ATP and an affinity label attached to rabbit muscle phosphofructokinase

The smallest enzymatically active form of rabbit muscle phosphofructokinase is a tetramer of four identical or nearly identical monomers. The enzyme is inhibited by ATP, and this inhibition by ATP is relieved by the activating adenine nucleotides adenosine cyclic 3',5'-phosphate, AMP, and ADP. Each monomer contains one binding site specific for the inhibitor ATP and another site specific for the activating adenine nucleotides. The enzyme can also be activated by covalently labeling the activating adenine nucleotide binding sites with the affinity label 5'-[p-(fluorosulfonyl)benzoyl]adenosine. These activator binding sites on the enzyme have been covalently labeled to various degrees, ranging from an average value of less than one label per tetramer to four labels per tetramer, and the free-energy coupling, delta Gxy, between the covalently bound affinity label and ATP binding at the inhibitory site was determined. For enzyme preparations containing four labels per tetramer, delta Gxy is approximately 1 kcal/mol at pH 6.95 and 25 degrees C. A very significant free-energy coupling is observed in those preparations containing an average of one label per tetramer and less, and the change in delta Gxy in going from native tetramers to ones containing an average of two labels per tetramer is twice as great as the change in delta Gxy observed in going from tetramers containing an average of two labels per tetramer to ones containing four labels per tetramer, suggesting that modification of the final two monomers in the tetramer contributes much less to the antagonistic effect on ATP binding than does modification of the first two monomers in the tetramer.     

Photochemical labeling of bovine pancreatic ribonuclease A with 8-azidoadenosine 3',5'-bisphosphate

A simple method has been developed for the preparation of 5'-32P-labeled 8-azidoadenosine 3',5'-bisphosphate (p8N3Ap) for use in photoaffinity labeling studies. Irradiation of a complex between p8N3Ap and bovine pancreatic ribonuclease A (RNase A) with light of 300-350 nm led to the covalent attachment of the nucleotide to the enzyme. RNase A could also be labeled in the dark with prephotolyzed p8N3Ap. In either case, the nucleotide reacted with the same tryptic peptide, encompassing amino acids 67-85 of the protein. The site of labeling was determined to be either Thr-78 or Thr-82, both of which are close to or at the pyrimidine binding site of the enzyme. This result is consistent with recent nuclear magnetic resonance and X-ray studies which indicate that 8-substituted adenine nucleotides interact with the pyrimidine binding site of RNase A.     

A rapid and efficient new method of purification of glutamate dehydrogenase by affinity chromatography on GTP-sepharose

The very high affinity for GTP of glutamate dehydrogenase was used to purify this enzyme by affinity chromatography. After periodic acid oxidation, GTP was covalently bound to an activated Sepharose. When crude mitochondrial extracts were applied on a column of this GTP-Sepharose, glutamate dehydrogenase was retained with very few other proteins. Glutamate dehydrogenase from rat liver was eluted with a KCl gradient with only one contaminating protein. From a pig heart mitochondrial extract the enzyme was purified 300-fold in one step. A chromatography on hydroxyapatite was sufficient to achieve the purification. This very simple technique avoids the long and troublesome crystallization steps generally involved in glutamate dehydrogenase purification.     

Affinity labeling of Escherichia coli DNA polymerase I by 5'-fluorosulfonylbenzoyladenosine. Identification of the domain essential for polymerization and Arg-682 as the site of reactivity

Preincubation of Escherichia coli DNA polymerase I (pol I) with 5'-fluorosulfonylbenzoyladenosine (5'-FSBA) results in an irreversible inactivation of DNA polymerase activity with concomitant covalent binding of 5'-FSBA to enzyme. pol I-associated 3'-5' exonuclease activity, however, remains unaffected. Kinetic studies of inactivation indicate that the degree of inactivation is directly proportional to the concentration of 5'-FSBA and increases linearly with time. The presence of the metal chelate form of dNTP substrates or template primer, but not the template or primer alone, protects the enzyme from inactivation by 5'-FSBA. A complete inactivation of polymerase activity occurs when 2 mol of 5'-FSBA are covalently linked to 1 mol of enzyme, suggesting two sites of modification. Tryptic peptide mapping of 5'-FSBA-treated enzyme revealed the presence of two distinct peptides containing the affinity label, confirming the presence of two reactive sites in the enzyme. However, we find that only one of the two sites is essential for the polymerase activity since, in the presence of substrate dNTP or template primer during preincubation of enzyme with 5'-FSBA, incorporation of the affinity label is reduced by only 1 mol. Peptide analysis of dNTP or template primer-protected enzyme further revealed that a peptide eluting at 35 min from the C-18 matrix was protected from the 5'-FSBA reaction. It is therefore concluded that this peptide contains the domain essential for polymerase activity. Staphylococcus aureus V-8 protease digestion, amino acid composition, and sequence analysis of this peptide revealed this domain to span residues 669 to 687 in the primary amino acid sequence of pol I, and arginine 682 was found to be the site of 5'-FSBA reactivity.     

Interaction of glycogen phosphorylase with 8-azidoadenosine 5'-monophosphate, a photoaffinity analog of AMP

The ability of 8-azidoadenosine 5'-monophosphate (N3AMP) to act as a photoaffinity label for the AMP binding site on glycogen phosphorylase (EC 2.4.1.1) was tested. 8-Azidoadenosine 5'-monophosphate can replace AMP as an allosteric modifier of both phosphorylases a and b; the pH optimum and the extent of activation are comparable to that observed with AMP. 8-Azidoadenosine 5'-monophosphate resembles the natural activator in having a higher affinity for phosphorylase a. The effects of 8-azidoadenosine 5'-monophosphate and AMP on phosphorylase b are additive when each is present at a concentration which gives less than 50% activation. Increasing the concentration of the substrate, glucose 1-phosphate, decreases the apparent activation constant (Ka) for the interaction of 8-azidoadenosine 5'-monophosphate with phosphorylase b. Glucose 6-phosphate is an inhibitor of phosphorylase b with either AMP or 8-azidoadenosine 5'-monophosphate. In the presence of ultraviolet light, 8-azidoadenosine 5'-monophosphate is irreversibly incorporated into phosphorylase a; incorporation at the allosteric site can be reduced if AMP is added prior to irradiation. Under the conditions used in the photolysis experiments, 3--5% of the available AMP sites were labeled with 8-azidoadenosine 5'-monophosphate. The data indicate the potential usefulness of 8-azidoadenosine 5'-monophosphate as a probe for the AMP site on phosphorylase.     

Catalytic competence, a new criterion for affinity labeling. Demonstration of the reversible enzymatic interconversion of estrone and estradiol-17 beta covalently bound to human placental estradiol-17 beta dehydrogenase.

Human placental estradiol-17beta dehydrogenase is rapidly inactivated upon treatment with 3-bromoacetoxyestrone. Pseudo-first order kinetic data are obtained and inactivation is accompanied by incorporation of 1 mol of 3-acetoxyestrone/mol of subunit (Mr =34,000). Treatment of the inactivated enzyme with (4S)-[4-2H]DPNH results in the formation of covalently bound [17alpha-2H]estradiol-17beta, which can be released by hydrolysis and identified by gas chromatography-mass sepctrometry. When (4R)-[4-2H]DPNH was used, deuterium was not transferred. Thus, the normal stereochemistry of hydridetransfer is preserved for both partners. After treatment with p-mercuribenzoate, affinity-labeled estradiol-17beta dehyrogenase is no longer able to caralyze reduction its covalently bound estrone; in the presence of DPNH and native enzyme, however, reduction occurs, demonstrating that affinity-labeled enzyme can itself serve as subtrate for native estradiol-17beta dehydrogenase. The reversible enzymatic interconversion of covalently bound estrone was demonstrated using a transhydrogenase assay. The ability of an enzyme to catalyze its normal reaction with a covalently bound substrate is termed catalytic competence, and is considered to be a new criterion for affinity labeling.     

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.     

Identification of the covalently bound flavin of thiamin dehydrogenase.

Thiamin dehydrogenase, a flavoprotein isolated from an unidentified soil bacterium, contains 1 mol of covalently bound FAD/mol of enzyme. A flavin peptide, isolated from tryptic-chymotryptic digests of the enzyme and hydrolyzed to the FMN level, shows a pH-dependent fluorescence yield being maximal at pH 3.5 to 4.0 and decreasing over 90% at pH 7.5 with a pKa of 5.8. Acid hydrolysis of the peptide results in an aminoacylflavin which shows a pKa of fluorescence quenching of 5.2. Absorption and electron paramagnetic resonance spectral data show the covalent substituent to be at the 8alpha position of the flavin as is the case with all known enzymes containing covalently bound flavin. The aminoacylflavin gives a negative Pauly reaction but yields 1 mol of histidine on drastic acid hydrolysis thus showing an imidazole ring nitrogen as the 8alpha substituent of the flavin. The aminoacylflavin differs from synthetic 8alpha-[N(3)-histidyl]riboflavin or its acid-modified form in pKa of fluorescence quenching, in electrophoretic mobility, in being reduced by borohydride, and in being labile to storage, yielding 8-formylriboflavin. In all of these properties, however, the 8alpha-histidylriboflavin isolated from thiamin dehydrogenase is indistinguishable from 8alpha-[N(1)-histidyl]riboflavin. It is therefore concluded that the FAD moiety of thiamin dehydrogenase is covalently linked via the 8alpha-methylene group to the N(1) position of the imidazole ring of histidine.     

Total number and differentiation of nucleotide binding sites on mitochondrial F1-ATPase. An approach by photolabeling and equilibrium binding studies

In this study 3'-O-[3-(4-azido-2-nitrophenyl)propionyl]-ADP was used as a photoaffinity analog for nucleotide binding sites on nucleotide-depleted F1-ATPase. Catalytic and binding properties of the labeled enzyme were investigated. The analog behaves as a competitive inhibitor in the dark (Ki = 50 microM). Photoirradiation of F1 in the presence of the analog leads to inactivation depending linearly on the incorporation of label. Complete inactivation is achieved at a stoichiometry of 3 mol/mol F1. The label is distributed between alpha and beta subunits in a ratio of 30%:70%. Although three sites were blocked covalently by photolabeling, three reversible sites of much higher affinity than the labeled sites were preserved. Mild alkaline treatment of photoinactivated enzyme leads to almost complete reactivation which is due to hydrolysis of the 3'-ester bond and release of the ADP moiety from the covalently bound analog. The conclusions drawn are as follows. The total number of sites which can be simultaneously occupied by nucleotides on F1 is six. Adopting the finding [Grubmeyer, C. & Penefsky, H. S. (1981) J. Biol. Chem. 256, 3718-3727] that the high-affinity sites are the catalytic ones which can be covalently labeled by 3'-O-[5-azidonaphthoyl(1)]-ADP [Lübben, M., Lücken, U., Weber, J. & Sch?fer, G. (1984) Eur. J. Biochem. 143, 483-490], it appears likely that azidonitrophenylpropionyl-ADP is a specific photolabel for the lower-affinity sites on nucleotide-depleted F1. This means that both types of sites can be differentiated by specific photoaffinity analogs. The labeled low-affinity sites interact with the catalytic sites, abolishing enzyme turnover, when steadily occupied by ADP kept in place by the covalently linking residue, which by itself has no inhibitory effect on the enzyme.     

Affinity labeling of phosphoglycerate kinase by 5'-[p-(fluorosulfonyl)benzoyl]-1,N6-ethenoadenosine

Yeast phosphoglycerate kinase is irreversibly inactivated upon incubation with 5'-[p-(fluorosulfonyl)-benzoyl]-1-N6-ethenoadenosine (5'-FSB epsilon A), an analogue to the nucleotide substrate. Marked protection against inactivation occurs with MgATP, ATP, MgADP, ADP, and 3-phosphoglycerate, suggesting that a part of the catalytic center is modified. The time dependence of the inactivation is characterized by a nonlinear kinetic profile. Curve fitting of various models for ligand binding to the enzyme suggested a two-site model. Modification of one of the sites appears to protect the catalytically essential site from modification. Stoichiometric studies show that the relationship between moles of 5'-FSB epsilon A incorporated per mole of enzyme and the residual enzymatic activity also shows nonlinear behavior. An extrapolated value of 1.5 mol of bound label/mol of enzyme corresponds to complete inactivation. The apparent overall pseudo first-order rate constant for the reaction between phosphoglycerate kinase and 5'-FSB epsilon A, as well as the separate rate constants for the modification, exhibit saturation behavior with respect to the concentration of 5'-FSB epsilon A, indicative of a rapid reversible binding of the reagent to the enzyme prior to modification.