Inactivation of Escherichia coli glycerol kinase by 5'-[p-(fluorosulfonyl)benzoyl]adenosine: protection by the hydrolyzed reagent

Incubation of Escherichia coli glycerol kinase (EC 2.7.1.30; ATP:glycerol 3-phosphotransferase) with 5'-[p-(fluorosulfonyl)benzoyl]adenosine (FSO2BzAdo) at pH 8.0 and 25 degrees C results in the loss of enzyme activity, which is not restored by the addition of beta-mercaptoethanol or dithiothreitol. The FSO2BzAdo concentration dependence of the inactivation kinetics is described by a mechanism that includes the equilibrium binding of the reagent to the enzyme prior to a first-order inactivation reaction in addition to effects of reagent hydrolysis. The hydrolysis of the reagent has two effects on the observed kinetics. The first effect is deviation from pseudo-first-order kinetic behavior due to depletion of the reagent. The second effect is the novel protection of the enzyme from inactivation due to binding of the sulfonate hydrolysis product. The rate constant for the hydrolysis reaction, determined independently from the kinetics of F- release, is 0.021 min-1 under these conditions. Determinations of the reaction stoichiometry with 3H-labeled FSO2BzAdo show that the inactivation is associated with the covalent incorporation of 1.08 mol of reagent/mol of enzyme subunit. Ligand protection experiments show that ATP, AMP, dAMP, NADH, 5'-adenylyl imidodiphosphate, and the sulfonate hydrolysis product of FSO2BzAdo provide protection from inactivation. The protection obtained with ATP is not dependent on Mg2+. Less protection is obtained with glycerol, GMP, etheno-AMP, and cAMP. No protection is obtained with CMP, UMP, TMP, etheno-CMP, GTP, or fructose 1,6-bisphosphate. The results are consistent with modification by FSO2BzAdo of a single adenine nucleotide binding site per enzyme subunit.     

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.     

Mapping of the ATP-binding domain of human fructosamine 3-kinase-related protein by affinity labelling with 5'-[p-(fluorosulfonyl)benzoyl]adenosine

The modification of proteins by reducing sugars through the process of non-enzymatic glycation is one of the principal mechanisms by which hyperglycaemia may precipitate the development of diabetic complications. Fn3K (fructosamine 3-kinase) and Fn3KRP (Fn3K-related protein) are two recently discovered enzymes that may play roles in metabolizing early glycation products. However, although the activity of these enzymes towards various glycated substrates has been established, very little is known about their structure-function relationships or their respective mechanisms of action. Furthermore, their only structural similarities noted to date with members of other kinase families has been with the bacterial aminoglycoside kinases. In the present study, we employed affinity labelling with the ATP analogue FSBA {5'-p-[(fluorosulfonyl)benzoyl]adenosine} to probe the active-site topology of Fn3KRP as an example of this enigmatic family of kinases. FSBA was found to modify Fn3KRP at five distinct sites; four of these were predicted to be localized in close proximity to its ATP-binding site, based on alignments with the aminoglycoside kinase APH(3')-IIIa, and examination of its published tertiary structure. The results of the present studies provide evidence that Fn3KRP possesses an ATP-binding domain that is structurally related to that of both the aminoglycoside kinases and eukaryotic protein kinases.     

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.     

Rabbit muscle phosphofructokinase. 2. Inactivation by the affinity label 5'-[p-(fluorosulfonyl)benzoyl]-1,N6-ethenoadenosine

The reaction of the fluorescent affinity label 5'-[p-(fluorosulfonyl)benzoyl]-1,N6-ethenoadenosine with rabbit skeletal muscle phosphofructokinase results in an inactivation of the enzyme and in the covalent incorporation of up to one label/monomer. The substrates, MgATP and fructose 6-phosphate, each protect against inactivation of the enzyme, but neither diminishes the extent of covalent incorporation of the label, indicating that the inactivation is not the result of covalent incorporation of the label. Dithiothreitol reactivates the inactivated enzyme but does not reduce the extent of incorporation of the label. A determination of the number of free sulfhydryl groups on the enzyme as a function of the extent of inactivation by the reagent suggests that the inactivation is associated with the loss of two free sulfhydryl groups per phosphofructokinase monomer. The inactivation reaction appears to involve the reversible formation of an enzyme-reagent complex (Kd = 1.11 mM) prior to the conversion of the complex to inactive enzyme (k1 = 0.98 min-1). In view of the protection afforded by either substrate and the evidence suggesting the formation of an enzyme-reagent complex prior to inactivation, it would appear that the inactivation results from a reagent-mediated formation of a disulfide bond between two cysteinyl residues in close proximity, possibly in or near the catalytic site of the enzyme. The site of covalent attachment of the label appears to be the binding site specific for the activating adenine nucleotides cAMP, AMP, and ADP. The extent of covalent incorporation of the label at this site is diminished in the presence of cAMP, and phosphofructokinase modified at this site by this affinity label is no longer subject to activation by cAMP.     

Affinity labeling of rabbit muscle fructose-1,6-bisphosphate aldolase with 5'-[p-(fluorosulfonyl)benzoyl]-1,N6-ethenoadenosine

Aldolase contains one tight binding site and one weak binding site per subunit for ATP [Kasprzak, A. and Kochman, M. (1980) Eur. J. Biochem. 104, 443-450]. The reaction of the ATP analog 5'-[p-(fluorosulfonyl)benzoyl]-1,N6-ethenoadenosine with rabbit aldolase A results in linear inactivation of enzyme with respect to covalent linkage of fluorescent label. The enzyme is completely protected against modification in the presence of saturating covalent binding (k2 = 0.033 min-1) is preceded by a fast reversible binding step (Ki = 6.8 mM). Chemical modification of aldolase leads to formation of stable N epsilon (4-carboxybenzenesulfonyl-lysine (Cbs-Lys) and O-(4-carboxybenzenesulfonyl-tyrosine (Cbs-Tyr) derivatives. Almost all Cbs-Lys was found in the N-terminal CNBr peptide (CN-1), whereas Cbs-Tyr was present both in the N-terminal (CN-1) and C-terminal (CN-2) peptide. From carboxypeptidase digestion and tryptic peptide analysis, Cbs-Lys was localized in position 107, a small part of Cbs-Tyr was detected in position 84, and the majority of Cbs-Tyr was found in the C-terminal position Tyr-363. We conclude that the covalent binding of the ATP analog occurs at the mononucleotide tight-binding site of aldolase and is associated with modification of Lys-107 and Tyr-363. This conclusion is based on the measurements of enzymatic activity loss as a function of ATP analog incorporation as well as on previous data. It is postulated that Lys-107, which is the C-6 phosphate binding site for fructose-1,6-P2, is in close proximity to the functionally important Tyr-363. The rather small extent of modification of Tyr-84 (0.15 mol/subunit), is due either to nonspecific protein modification or labeling of the weak mononucleotide binding site.     

Identification of the ATP binding sites of the carbamyl phosphate synthetase domain of the Syrian hamster multifunctional protein CAD by affinity labeling with 5'-[p-(fluorosulfonyl)benzoyl]adenosine.

The ATP analogue 5'-[p-(fluorosulfonyl)benzoyl]adenosine (FSBA) was used to chemically modify the ATP binding sites of the carbamyl phosphate synthetase domain of CAD, the multifunctional protein that catalyzes the first steps in mammalian pyrimidine biosynthesis. Reaction of CAD with FSBA resulted in the inactivation of the ammonia- and glutamine-dependent CPSase activities but had no effect on its glutaminase, aspartate transcarbamylase, or dihydroorotase activities. ATP protected CAD against inactivation by FSBA whereas the presence of the allosteric effectors UTP and PRPP afforded little protection, which suggests that the ATP binding sites were specifically labeled. The inactivation exhibited saturation behavior with respect to FSBA with a K1 of 0.93 mM. Of the two ATP-dependent partial activities of carbamyl phosphate synthetase, bicarbonate-dependent ATPase was inactivated more rapidly than the carbamyl phosphate dependent ATP synthetase, which indicates that these partial reactions occur at distinct ATP binding sites. The stoichiometry of [14C]FSBA labeling showed that only 0.4-0.5 mol of FSBA/mol of protein was required for complete inactivation. Incorporation of radiolabeled FSBA into CAD and subsequent proteolysis, gel electrophoresis, and fluorography demonstrated that only the carbamyl phosphate synthetase domain of CAD is labeled. Amino acid sequencing of the principal peaks resulting from tryptic digests of FSBA-modified CAD located the sites of FSBA modification in regions that exhibit high homology to ATP binding sites of other known proteins. Thus CAD has two ATP binding sites, one in each of the two highly homologous halves of the carbamyl phosphate domain which catalyze distinct ATP-dependent partial reactions in carbamyl phosphate synthesis.     

Mitochondrial nicotinamide nucleotide transhydrogenase: nonidentical modification by N,N'-dicyclohexylcarbodiimide and N-(ethoxycarbonyl)-2-ethoxy-1,2-dihydroquinoline at the NAD(H) binding site

The energy-linked nicotinamide nucleotide transhydrogenase (TH) purified from bovine heart mitochondria is inhibited by the carboxyl group modifiers, N,N'-dicyclohexylcarbodiimide (DCCD) and N-(ethoxycarbonyl)-2-ethoxy-1,2-dihydroquinoline (EEDQ). With either reagent, complete activity inhibition corresponds to modification of one carboxyl group per 2 mol (monomers) of this dimeric enzyme, suggesting half-site reactivity toward DCCD and EEDQ [D. C. Phelps, and Y. Hatefi (1984) Biochemistry 23, 4475-4480; 6340-6344]. It has also been shown in the former reference that DCCD appears to modify TH at the NAD(H)-binding site. The present paper presents data suggesting that EEDQ also binds at or near the NAD(H)-binding domain of TH, but at a site not identical to that of DCCD: TH modified with and inhibited approximately 85% by EEDQ could be further labeled with [14C]DCCD to the extent of 70% of the maximum in the same time period that unmodified TH was modified by [14C]DCCD to near saturation (1 mol DCCD/TH dimer); DCCD-modified TH did not bind to NAD-agarose, while EEDQ-modified TH showed partial affinity for NAD-agarose; 5'-AMP completely protected TH against modification by DCCD, but showed only a weak protective effect against EEDQ; by contrast, NMNH, which is a TH substrate and binds to the NADH site, did not protect TH against DCCD, but completely protected the enzyme against attack by EEDQ. The results are consistent with the possibility that DCCD modifies TH where the 5'-AMP moiety of NAD(H) binds, while EEDQ modifies the enzyme where the NMN(H) moiety of NAD(H) resides.     

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.     

Irreversible inhibition of human natural killer cell natural cytotoxicity by modification of the extracellular membrane by the adenine nucleotide analog 5′-p-(fluorosulfonyl)benzoyl adenosine

Extracellular adenine nucleotides are inhibitors of the human natural killer cell line NK3.3 natural cytotoxicity activity. Natural cytotoxicity was inhibited approximately 26% by 1 mM ATP and 21% by 1 mM ADP. 5′-Adenylyl imidodiphosphate, a nonhydrolyzable ATP analog, inhibited natural cytotoxicity by 41% at a concentration of 1 mM and > 97% at a concentration of 10 mM. In contrast, AMP was not inhibitory. Adenosine was a weak inhibitor of natural cytotoxicity and may represent an alternate regulatory pathway. Removal of the nucleotides resulted in the restoration of control levels of natural cytotoxicity activity. The affinity label 5′-p-(fluorosulfonyl)benzoyladenosine (5′-FSBA) is a synthetic analog of ATP or ADP containing an electrophilic fluorosulfonyl group capable of covalently modifying proteins at adenine di- and triphosphate nucleotide-binding sites. Natural cytotoxicity was irreversibly inhibited by modification of the extracellular membrane of NK3.3 cells by 5′-FSBA. This inhibition was concentration dependent with an I₅₀ ∼ 100 μM and complete inhibition at 1 mM. Modification of NK3.3 by 5′-FSBA did not affect the formation of effector—target cell conjugates; however, granule release was inhibited. This targets the site of inhibition by 5′-FSBA modification to a pathway preceding granule release. Irreversible, covalent modification of surface adenine nucleotide-binding proteins by 5′-FSBA provides a probe to study the role of specific adenine nucleotide-binding proteins in the extracellular regulation of natural killer cytolytic activity by adenine nucleotides.     

Chemical modification of Salmonella typhimurium phosphoribosylpyrophosphate synthetase with 5'-(p-fluorosulfonylbenzoyl)adenosine. Identification of an active site histidine

Liquid chromatographic procedures have been developed for rapidly locating the site of reaction of chemical modification reagents with Salmonella typhimurium 5-phosphoribosyl-alpha-1-pyrophosphate (PRPP) synthetase. The enzyme was reacted with the active site-directed reagent 5'-(p-fluorosulfonylbenzoyl)adenosine (FSBA). FSBA bound to the enzyme with an apparent KD of 1.7 +/- 0.4 mM. The enzyme was inactivated during the reaction, and a limiting stoichiometry of 1.2 mol of FSBA/mol of enzyme subunit corresponded to complete inactivation. Inclusion of ATP in the reaction protected the enzyme from inactivation and incorporation of the reagent. Inclusion of ribose 5-phosphate increased the rate of reaction of PRPP synthetase with FSBA. Amino acid analyses of acid hydrolysates of modified enzyme failed to detect any known FSBA-amino acid adducts. Tryptic digestion of 5'-(p-fluorosulfonylbenzoyl)-[3H]adenosine-modified enzyme at pH 7.0 yielded a single radioactive peptide. The peptide, TR-1, was subjected to combined V8 and Asp-N protease digestion, and a single radioactive peptide was isolated. This radioactive peptide yielded the sequence Asp-Leu-His-Ala-Glu, which corresponded to amino acid residues 128-132 in S. typhimurium PRPP synthetase. No radioactivity was associated with any of the phenylthiohydantoin-amino acid fractions, all of which were recovered in good yield. A majority of the radioactivity was found in the waste effluent (64%) and on the glass fiber filter loaded into the sequenator (23%). The lability of the modification and the sequence of this peptide indicate His130 as the site of reaction with FSBA.     

Mitochondrial nicotinamide nucleotide transhydrogenase: inhibition by ethoxyformic anhydride, dansyl chloride, and pyridoxal phosphate

Mitochondrial energy-linked nicotinamide nucleotide transhydrogenase (TH; EC 1.6.1.1) was inactivated by treatment with pyridoxal phosphate, ethoxyformic anhydride (EFA) or dansyl chloride. NADP and NADPH, but not NAD and NADH, protected TH against inhibition by pyridoxal phosphate, and L-lysine reversed this inhibition. The results suggested modification of an essential lysyl residue by pyridoxal phosphate, possibly at the NADP(H) binding site of TH. EFA and dansyl chloride inhibited TH in a similar manner. The effect of pH on the rate of inhibition of TH by EFA and dansyl chloride was the same, and in both cases addition of NADP and particularly NADPH accelerated the rate of inhibition, while addition of NAD or NADH had no effect. Double inhibition studies, using in one experiment dithiothreitol-reversible inhibition by 5,5'-dithiobis(2-nitrobenzoic acid) to protect the thiol groups of TH, and in another experiment lysine-reversible inhibition by pyridoxal phosphate to protect the putative essential lysyl residues of the enzyme, followed in each case by further treatment of the protected TH with EFA or dansyl chloride, suggested that the inhibitions by EFA and dansyl chloride were independent of the inhibitions by 5,5'-dithiobis (2-nitrobenzoic acid) and pyridoxal phosphate. The inhibitors discussed above are interesting, because pyridoxal phosphate is the only reagent known which appears to modify an essential residue in the NADP(H), but not the NAD(H), binding site of TH, and EFA and dansyl chloride are the only inhibitors known which appear to react with essential residues outside the active site of TH. It is possible that EFA and dansyl chloride inhibitions involve modification of essential prototropic residues in the proton translocation domain of the enzyme.     

Mitochondrial nicotinamide nucleotide transhydrogenase: NADPH binding increases and NADP binding decreases the acidity and susceptibility to modification of cysteine-893

The mitochondrial nicotinamide nucleotide transhydrogenase is a dimeric enzyme of monomer Mr 110,000. It is located in the inner mitochondrial membrane and catalyzes hydride ion transfer between NAD(H) and NADP(H) in a reaction that is coupled to proton translocation across the inner membrane. The amino acid sequence and the nucleotide binding sites of the enzyme have been determined [Yamaguchi, M., Hatefi, Y., Trach, K., & Hoch, J.A. (1988) J. Biol. Chem. 263, 2761-2767; Wakabayashi, S., & Hatefi, Y. (1987) Biochem. Int. 15, 915-924]. N-Ethylmaleimide, as well as other sulfhydryl group modifiers, inhibits the transhydrogenase. The presence of NADP in the incubation mixture suppressed the inhibition rate by N-ethylmaleimide, and the presence of NADPH greatly increased it. NAD and NADH had little or no effect. The NADPH effect was concentration dependent and saturable, with a half-maximal NADPH concentration effect close to the Km of the enzyme for NADPH. Study of the effect of pH on the N-ethylmaleimide inhibition rate showed that NADPH binding by the enzyme lowers the apparent pKa of the N-ethylmaleimide-sensitive group by 0.4 of a pH unit and NADP binding raises this pKa by 0.4 of a pH unit, thus providing a rationale for the effects of NADP and NADPH on the N-ethylmaleimide inhibition rate. With the use of N-[3H]ethylmaleimide, the modified sulfhydryl group involved in the NADP(H)-modulated inhibition of the transhydrogenase was identified as that belonging to Cys-893, which is located 113 residues upstream of the tyrosyl residue modified by [p-(fluorosulfonyl)benzoyl]-5'-adenosine at the putative NADP(H) binding site of the enzyme (see above references).(ABSTRACT TRUNCATED AT 250 WORDS)     

Energy-linked nicotinamide nucleotide transhydrogenase: hydrodynamic properties and active form of purified and membrane-bound mitochondrial transhydrogenase from beef heart

The mitochondrial nicotinamide nucleotide transhydrogenase from beef heart was investigated with respect to minimal assembly of the purified enzyme and of the enzyme in the mitochondrial inner membrane. Studies of the hydrodynamic properties of the purified enzyme in the presence of 0.3% Triton X-100 allowed determination of the Stokes radius, sedimentation constant, partial specific volume, frictional ratio, and molecular weight. Under these conditions transhydrogenase existed as an inactive monomer, suggesting that monomerization may be accompanied by inactivation. Radiation inactivation was used to determine the functional molecular size of purified detergent-dispersed transhydrogenase and transhydrogenase in beef heart submitochondrial particles. Under these conditions the catalytic activity of both the purified and the membrane-bound enzyme was found to be catalyzed by a dimeric form of the enzyme. These results suggest for the first time that the minimal functional assembly of detergent-dispersed as well as membrane-bound transhydrogenase is a dimer, which is not functionally associated with, for example, complex I or ATPase. In addition, the results are consistent with the possibility that the two subunits of transhydrogenase are catalytically active in an alternating fashion according to a previously proposed half-of-the-sites reactivity model.     

Conformational diversity in NAD(H) and interacting transhydrogenase nicotinamide nucleotide binding domains

Transhydrogenase (TH) couples direct and stereospecific hydride transfer between NAD(H) and NADP(H), bound within soluble domains I and III, respectively, to proton translocation across membrane bound domain II. The cocrystal structure of Rhodospirillum rubrum TH domains I and III has been determined in the presence of limiting NADH, under conditions in which the subunits reach equilibrium during crystallization. The crystals contain three heterotrimeric complexes, dI(2)dIII, in the asymmetric unit. Multiple conformations of loops and side-chains, and NAD(H) cofactors, are observed in domain I pertaining to substrate/product exchange, and highlighting electrostatic interactions during the hydride transfer. Two interacting NAD(H)-NADPH pairs are observed where alternate conformations of the NAD(H) phosphodiester and conserved arginine side-chains are correlated. In addition, the stereochemistry of one NAD(H)-NADPH pair approaches that expected for nicotinamide hydride transfer reactions. The cocrystal structure exhibits non-crystallographic symmetry that implies another orientation for domain III, which could occur in dimeric TH. Superposition of the "closed" form of domain III (PDB 1PNO, chain A) onto the dI(2)dIII complex reveals a severe steric conflict of highly conserved loops in domains I and III. This overlap, and the overlap with a 2-fold related domain III, suggests that motions of loop D within domain III and of the entire domain are correlated during turnover. The results support the concept that proton pumping in TH is driven by the difference in binding affinity for oxidized and reduced nicotinamide cofactors, and in the absence of a difference in redox potential, must occur through conformational effects.     

Rabbit muscle phosphofructokinase. Modification of molecular and regulatory kinetic properties with the affinity label 5'-p-(fluorosulfonyl)benzoyl adenosine.

The affinity label 5'-p-(fluorosulfonyl)benzoyl adenosine modifies rabbit muscle phosphofructokinase to the extent of one group/subunit. Modification appears to occur at a binding site specific for AMP, cyclic AMP, and ADP, i.e. those adenine nucleotides which are activators under conditions where regulatory kinetic behavior is obtained. The consequences of the modification are consistent with the model proposed previously for correlation between the pK of specific ionizable groups, regulatory kinetic behavior, ligand binding, and the reversible cold inactivation of the enzyme (Frieden, C., Gilbert. H. R., and Bock, P. E. (1976) J. Biol. Chem. 251, 5644-5647). Thus, the modification shifts the apparent pK of the essential ionizable groups from 6.9 to 6.4 at 25 degrees C, with the result that regulatory kinetic behavior at pH 6.9 and 25 degrees C is lost. Furthermore, the apparent affinity of a site (other than the active site) for ATP, as measured by ATP-dependent quenching of intrinsic protein fluorescence at pH 6.9 and 25 degrees C, is decreased by the modification. Regulatory kinetic behavior for both substrates is obtained with the modified enzyme at a lower pH, consistent with the downward shift in the pK of the ionizable groups, but sensitivity to cAMP activation is abolished by the modification. The loss of regulatory kinetic behavior upon modification of sulfhydryl groups does not appear to be the same as that due to modification by the affinity label.     

Affinity chromatography and binding studies on immobilized 5'-monophosphate and adenosine 2',5'-bisphosphate of nicotinamide nucleotide transhydrogenase from Pseudomonas aeruginosa.

1. Nicotinamide nucleotide transhydrogenase from Pseudomonas aeruginosa was purified to apparent homogeneity with an improved method employing affinity chromatography on N6-(6aminohexyl)-adenosine 2', 5'-bisphosphate-Sepharose 4B. 2. Polyacrylamide gel electrophoresis of the purified transhydrogenase carried out in the presence of sodium dodecyl sulphate, indicated a minimal molecular weight of 55000 +/- 2000. 3. The kinetic and regulatory properties of the purified transhydrogenase resembled those of the crude enzyme, i.e., NADPH, adenosine 2'-monophosphate and Ca2+ were activators whereas NADP+ was inhibitory. 4. Nicotinamide nucleotide-specific release of binding of the transhydrogenase to N6-(6-aminohexyl)-adenosine-2',5'-bisphosphate-Sepharose and N6-(-aminohexyl)-adenosine-5'-monophosphate-Sepharose suggests the presence of at least two separate binding sites for nicotinamide nucleotides, one that is specific for NADP(H) and one that binds both NAD(H) and NADP(H). 5. Binding of transhydrogenase to N6-)6-aminohexyl)-adenosine-2',5'-bisphosphate-Sepharose and activation of the enzyme by adenosine-2',5'-bisphophate showed a marked pH dependence. In contrast, inhibition of the Ca2+-activated enzyme by adenosine 2',5'-bisphosphate was virtually constant at various pH values. This descrepancy was interpreted to indicate the existence of separate nucleotide-binding effector and active sites.     

Irreversible inhibition of adenylate cyclase by 5'-(p-bromomethylbenzoyl) adenosine

The effect of 5'-(p-bromomethylbenzoyl) adenosine (pBMBA) on adenylate cyclase from bovine caudate nucleus membranes was studied. Adenylyl-5'-methylenediphosphonate (but not adenosine) protected adenylate cyclase against inactivation by this compound. The degree of pBMBA-induced inhibition of adenylate cyclase increased in the presence of Mg2+. 5'-(p-fluorosulfonylbenzoyl) adenosine (pFSBA) was also a specific irreversible inhibitor of adenylate cyclase. It was demonstrated that the enzyme inactivated by pFSBA completely restored its activity under the action of dithiothreitol. The results obtained are indicative of the presence of the -SH group in the enzyme active site.     

Synthesis and properties of diastereoisomers of adenosine 5'-(O-1-thiotriphosphate) and adenosine 5'-(O-2-thiotriphosphate).

The chemical synthesis of adenosine 5'-(O-1-thiotriphosphate) (ATPalphaS) and adenosine 5'-(O-2-thiotriphosphate) (ATPbetaS) is described. Both exist as a pair of diastereomers, A and B. The isomers of ATPalphaS can be distinguished on the basis of their different reaction rates with myokinase as well as nucleoside diphosphate kinase. With both enzymes, isomer A reacts fast whereas isomer B reacts considerably more slowly. Phosphorylation of a mixture of isomers of ADPalphaS with pyruvate or acetate kinase yields ATPalphaS, isomer A, whereas the phosphoryl transfer with creatine or arginine kinase yields isomer B. The isomers of ATPbetaS differ in their reactivity with myosin. Isomer A is readily hydrolyzed, whereas isomer B is not. However, isomer B reacts faster with nucleoside diphosphate kinase and ADP than isomer A. Phosphoryl transfer with pyruvate kinase onto ADPbetaS yields ATPbetaS, isomer A, with acetate kinase, isomer B.     

Differential reactivity in the processing of [p-(halomethyl)benzoyl] formates by benzoylformate decarboxylase, a thiamin pyrophosphate dependent enzyme

A series of [p-(halomethyl)benzoyl]formates have been investigated as substrates for benzoylformate decarboxylase. These analogues vary from acting as normal substrates to acting as potent competitive inhibitors. The fluoro analogue is a substrate with Km (190 microM) and turnover number (20 s-1) similar to those of benzoylformate (Km = 340 microM; 81 s-1). The bromo analogue is a competitive inhibitor (Ki = 0.3 microM) and exhibits processing to eliminate bromide and form (p-methylbenzoyl)thiamin pyrophosphate. This modified cofactor hydrolyzes to form the p-methylbenzoate in quantitative yield. The chloro analogue [Km(app) = 21 microM] partitions between these two pathways such that 0.6% of the analogue ultimately forms p-methylbenzoate. These data are consistent with the interpretation that the leaving group potential of the halogen determines the enzymic fate of the analogue and that the potent inhibition observed for the bromo analogue is due to covalent modification of the cofactor.