Affinity labelling of the active site of brain phosphatidylinositol 4-kinase with 5'-fluorosulphonylbenzoyl-adenosine.

5'-p-Fluorosulphonylbenzoyl-adenosine (FSO2BzAdo), an affinity labelling analogue of ATP, was used to label the active site of sheep brain phosphatidylinositol 4-kinase (PtdIns 4-kinase). The incubation of PtdIns 4-kinase with concentrations of FSO2BzAdo as low as 50 microM resulted in considerate inactivation of the enzyme. (e.g. 55% less after 60 min with 50 microM FSO2BzAdo). The kinetics of inactivation of PtdIns 4-kinase by FSO2BzAdo suggest a two-step mechanism, in which a rapid reversible binding of FSO2BzAdo to the enzyme is followed by a covalent sulphonation step. The first-order rate constant (k2) for the inactivation of PtdIns 4-kinase was calculated to be 0.063 min-1, and the steady-state constant of inactivation (Ki) to be 200 microM. Preincubation of the enzyme with either ATP plus Mg2+, or PtdIns alone, prior to addition of FSO2BzAdo reduced the degree of inactivation of the enzyme; suggesting that FSO2BzAdo binds within the active site PtdIns 4-kinase. Moreover, since ATP plus Mg2+ provided the greatest protection against inactivation, it is concluded that the main site of labelling of PtdIns 4-kinase by FSO2BzAdo is within the ATP-binding site of the enzyme. Results obtained from chemical modification experiments, which employed pyridoxal 5'-phosphate and tetranitromethane, are consistent with a catalytically-essential lysine being present within the ATP-binding site of PtdIns 4-kinase. Therefore, it is hypothesised that the inactivation of PtdIns 4-kinase by FSO2BzAdo may be due to the labelling of this lysine residue.     

Affinity labeling of the active site of Escherichia coli glutamine synthetase by 5'-p-fluorosulfonylbenzoyladenosine

The interaction of Escherichia coli glutamine synthetase with the adenosine 5'-triphosphate analogue, 5'-p-fluorosulfonylbenzoyladenosine (5'-FSO2BzAdo), has been studied. This interaction results in the covalent attachment of the 5'-FSO2BzAdo to the enzyme with concomitant loss of catalytic activity. Although adenine nucleotides interact with glutamine synthetase at three distinct sites--a noncovalent AMP effector site, a regulatory site of covalent adenylylation, and the catalytic ATP/ADP binding site--our studies suggest that reaction with 5'-FSO2BzAdo occurs only at the active center. When glutamine synthetase was incubated with 5'-FSO2BzAdo, the decrease in catalytic activity obeyed pseudo-first order kinetics. The plot of the observed rate constant of inactivation versus the concentration of 5'-FSO2BzAdo was hyperbolic, consistent with reversible binding of the analogue to the enzyme prior to covalent attachment. Protection against inactivation was afforded by ATP and ADP; L-glutamate did not protect the enzyme against inactivation, but rather enhanced the rate of inactivation, consistent with the observations of others (Timmons, R. B., Rhee, S. G., Luterman, D. L., and Chock, P. B. (1974) Biochemistry 13, 4479-4485) that there is synergism in the binding of the two substrates to the enzyme. The incorporation of approximately 1.09 mol of the 5'-FSO2BzAdo/mol of glutamine synthetase subunit resulted in the total loss of enzymatic activity. The results suggest that 5'-FSO2BzAdo occupies the ATP binding site at the active center of glutamine synthetase and binds covalently to an amino acid residue nearby.     

Proximate sulfhydryl groups in the acetylglutamate complex of rat carbamylphosphate synthetase I: their reaction with the affinity reagent 5'-p-fluorosulfonylbenzoyladenosine

A preparation of rat carbamylphosphate synthetase I, isolated in the presence of antipain and stable without glycerol, has been used to investigate the effect of the allosteric activator, N-acetyl-L-glutamate (AcGlu), on the sulfhydryl chemistry of the enzyme. The enzyme X AcGlu complex was rapidly inactivated by several sulfhydryl group reagents and the ATP analog, 5'-p-fluorosulfonylbenzoyladenosine (FSO2BzAdo), with the loss of two sulfhydryl groups per monomer. Inactivation was much slower without AcGlu, and ATP/Mg2+/K+ provided complete protection. Reaction with a 1.1 molar excess of 4,4'-dipyridyldisulfide resulted in an intramonomer disulfide bond between groups that are probably juxtaposed in the activated enzyme, because 1.1 equivalents of the vicinal dithiol reagent, phenylarsine oxide, eliminated the rapid reaction with the disulfide. Evidence is presented that the same disulfide bond was formed in the reactions with 5-thiocyano-2-nitrobenzoic acid and FSO2BzAdo. Inactivation by FSO2BzAdo was a pseudo-first-order reaction. The concentration dependence of the rate is consistent with the reaction proceeding through a noncovalent complex (KI = 67 microM and k2 = 0.23 min-1 at pH 7.0, 30 degrees C). Protection from FSO2BzAdo by ATP required Mg2+ in excess of ATP with KMgATP = 4.5 microM at saturating free Mg2+ (0.1 M K+) and KMg2+ = 6.5 mM. KMgATP is close to Kd for the molecule of ATP that contributes the phosphoryl group of carbamylphosphate (H.B. Britton, V. Rubio, and S. Grisolia, (1979) Eur. J. Biochem. 102, 521-530]; KMg2+ agrees with the minimum value for the steady-state kinetic parameter, Ki,Mg2+, obtained under the same conditions. Dissociation constants for adenosine (320 microM), MgADP (110 microM) at 10 mM Mg2+, and AcGlu (100 microM) were also estimated.     

Inactivation of Escherichia coli glycerol kinase by 5,5'-dithiobis(2-nitrobenzoic acid) and N-ethylmaleimide: evidence for nucleotide regulatory binding sites

Glycerol kinase (EC 2.7.1.30, ATP:glycerol 3-phosphotransferase) from Escherichia coli is inactivated by 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) and by N-ethylmaleimide (NEM) in 0.1 M triethanolamine at pH 7 and 25 degrees C. The inactivation by DTNB is reversed by dithiothreitol. In the cases of both reagents, the kinetics of activity loss are pseudo first order. The dependencies of the rate constants on reagent concentration show that while the inactivation by NEM obeys second-order kinetics (k2app = 0.3 M-1 s-1), DTNB binds to the enzyme prior to the inactivation reaction; i.e., the pseudo-first-order rate constant shows a hyperbolic dependence on DTNB concentration. Complete inactivation by each reagent apparently involves the modification of two sulfhydryl groups per enzyme subunit. However, analysis of the kinetics of DTNB modification, as measured by the release of 2-nitro-5-thiobenzoate, shows that the inactivation is due to the modification of one sulfhydryl group per subunit, while two other groups are modified 6 and 15 times more slowly. The enzyme is protected from inactivation by the ligands glycerol, propane-1,2-diol, ATP, ADP, AMP, and cAMP but not by Mg2+, fructose 1,6-bisphosphate, or propane-1,3-diol. The protection afforded by ATP or AMP is not dependent on Mg2+. The kinetics of DTNB modification are different in the presence of glycerol or ATP, despite the observation that the degree of protection afforded by both of these ligands is the same.(ABSTRACT TRUNCATED AT 250 WORDS)     

Detection of the conformational change in the catalytic site of adenosine triphosphatase from beef liver mitochondria by affinity labeling with the dialdehyde derivative of ethenoadenosine triphosphate

Beef liver mitochondrial F1ATPase was inactivated by the 2',3'-dialdehyde derivative of ethenoATP (epsilon ATP) in a pseudo-first order reaction. The kinetics of protection of the enzyme against inactivation by various nucleoside triphosphates (NTPs) revealed that the dial-epsilon ATP was bound to the catalytic site as an affinity label. Certain anions (sulfate or bicarbonate) were ineffective for protection. In the early phase of the reaction, inactivation was due to the binding of 1 mol dial-epsilon ATP per mol enzyme. In this phase, dial-epsilon ATP bound exclusively to the subunit beta of the enzyme, indicating that the catalytic site is in this subunit. The fluorescence of the ethenoadenosine moiety, bound exclusively to the subunit beta of the enzyme, was measured as a conformational probe of the catalytic site region. Addition of ATP or CTP to the labeled enzyme resulted in a decrease in the fluorescence intensity. GTP and other NTPs were less effective than ATP or CTP. The anions (sulfate of bicarbonate) suppressed the ability of ATP to decrease the fluorescence in a competitive manner. Quantitative analysis of these fluorescence changes suggested that they might originate from the binding of the NTP to the regulatory site of the enzyme. These findings are in good agreement with the two-site model proposed by us (Wakagi, T. & Ohta, T. (1981) J. Biochem. 89, 1205) which was deduced from the steady state kinetics of the NTPase reactions catalyzed by the F1ATPase.     

Affinity labeling of catalytic subunit of bovine heart muscle cyclic AMP-dependent protein kinase by 5'-p-fluorosulfonylbenzoyladenosine.

Incubation of 5'-p-fluorosulfonylbenzoyladenosine with the catalytic subunit of bovine cardiac muscle cyclic AMP-dependent protein kinase led to the formation of an inactive enzyme irreversibly modified with approximately one mol of reagent per mol of subunit. The inactivation reaction followed pseudofirst order kinetics. The rate of inactivation at various reagent concentrations exhibited saturation kinetics implying that the reagent reversibly binds to the enzyme prior to inactivation. The addition of MgATP, MgADP, or MgAMP-PNP to the reaction mixture fully protected the enzyme from inactivation by 5'-p-fluorosulfonylbenzoyladenosine. The reagent was demonstrated to be a competitive inhibitor of MgATP with a Ki of 0.235 mM. Metal-free nucleotides were without effect upon the reaction rate while metal ions alone accelerated the inactivation rate up to 7-fold. The inclusion of casein or synthetic peptide substrate in the incubation mixture did not affect the reaction kinetics. Reaction of 5'-p-fluorosulfonylbenzoyladenosine with the kinase subunit exhibits all of the characteristics of affinity labeling of the MgATP-binding site.     

Affinity labeling of the catalytic and AMP allosteric sites of 3-hydroxy-3-methylglutaryl-coenzyme A reductase kinase by 5'-p-fluorosulfonylbenzoyladenosine

The nucleotide analogue 5'-p-fluorosulfonylbenzoyladenosine (FSBA) reacts irreversibly with rat liver cytosolic 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase kinase, causing a rapid loss of the AMP activation capacity and a slower inactivation of the catalytic activity. The rate constant for loss of AMP activation is about 10 times higher (kappa 1 = 0.112 min-1) than the rate constant of inactivation (kappa 2 = 0.0106 min-1). There is a good correspondence between the time-dependent inactivation of reductase kinase and the time-dependent incorporation of 5'-p-sulfonylbenzoyl[14C]adenosine ([14C]SBA). An average of 1.65 mol of reagent/mol of enzyme subunit is bound when reductase kinase is completely inactivated. The time-dependent incorporation is consistent with the postulate that covalent reaction of 1 mol of SBA/mol of subunit causes complete loss of AMP activation, whereas reaction of another mole of SBA/mol of subunit would lead to total inactivation. Protection against inactivation by the reagent is provided by the addition of Mg2+, AMP, Mg-ATP, or Mg-AMP to the incubation mixtures. In contrast, addition of ATP, 2'-AMP, or 3'-AMP has no effect on the rate constants. Mg-ATP protects preferentially the catalytic site against inactivation, whereas Mg-AMP at low concentration protects preferentially the allosteric site. Mg-ADP affords less protection than Mg-AMP to the allosteric site when both nucleotides are present at a concentration of 50 microM with 7.5 mM Mg2+. Experiments done with [14C]FSBA in the presence of some protectants have shown that a close correlation exists between the pattern of protection observed and the binding of [14C]SBA. The postulate is that there exists a catalytic site and an allosteric site in the reductase kinase subunit and that Mg-AMP is the main allosteric activator of the enzyme.     

2-[(4-Bromo-2,3-dioxobutyl)thio]adenosine 5'-monophosphate, a new nucleotide analogue that acts as an affinity label of pyruvate kinase

A new reactive adenine nucleotide has been synthesized: 2-[(4-bromo-2,3-dioxobutyl)thio]-adenosine 5'-monophosphate (2-BDB-TAMP). Adenosine 5'-monophosphate 1-oxide was synthesized by reaction of AMP with m-chloroperoxybenzoic acid. Treatment with NaOH followed by reaction with carbon disulfide yielded 2-thioadenosine 5'-monophosphate (TAMP). The final product was generated by reaction of TAMP with 1,4-dibromobutanedione. The structure of 2-BDB-TAMP was determined by UV, 1H NMR, and 13C NMR spectroscopy as well as by bromide and phosphorus analysis. Rabbit muscle pyruvate kinase is inactivated by 2-BDB-TAMP at pH 7.0 and 25 degrees C. The inactivation rate exhibits a nonlinear dependence on the reagent concentration with KI = 0.57 mM. Protection against inactivation is provided by ADP and ATP, in the presence of Mn2+, as well as by phosphoenolpyruvate, in the presence of K+; in addition, partial protection is provided by AMP plus Mn2+. Incubation of pyruvate kinase with 0.075 mM 2-BDB-TAMP for 70 min in the absence of protective ligands leads to incorporation of 1.55 mol of reagent/mol of enzyme subunit when the enzyme is 53% inactive. In the presence of ADP and Mn2+, only 0.96 mol of reagent/mol of subunit is incorporated at 70 min, while the enzyme retains 100% activity. Similar results were obtained in the presence of ATP plus Mn2+. Assuming that the groups modified in the absence of ligands include those modified in the presence of the nucleotides, the 53% inactivation can be attributed to the modification of 0.59 (1.55-0.96) group per enzyme subunit.(ABSTRACT TRUNCATED AT 250 WORDS)     

Chemical modification of thiol groups of mitochondrial F1-ATPase from the yeast Schizosaccharomyces pombe. Involvement of alpha- and gamma-subunits in the enzyme activity

Mitochondrial F1-ATPase from the yeast Schizosaccharomyces pombe has been prepared under a stable form and in relatively high amounts by an improved purification procedure. Specific chemical modification of the enzyme by the thiol reagent N-ethylmaleimide (NEM) at pH 6.8 leads to complete inactivation characterized by complex kinetics and pH dependence, indicating that several thiols are related to the enzyme activity. A complete protection against NEM effect is afforded by low concentrations of nucleotides in the presence of Mg2+, with ADP and ATP being more efficient than GTP. A total binding of 5 mol of [14C]NEM/mol of F1-ATPase is obtained when the enzyme is 85% inactivated: 3 mol of the label are located on the alpha-subunits and 2 on the gamma-subunit. Two out of the 3 mol on the alpha-subunits bind very rapidly before any inactivation occurs, indicating that the two thiols modified are unrelated to the inactivation process. Complete protection by ATP against inactivation by NEM prevents the modification of three essential thiols out of the group of five thiols labeled in the absence of ATP: one is located on a alpha-subunit and two on the gamma-subunit. These two essential thiols of the gamma-subunit can be differentiated by modification with 6,6'-dithiodinicotinic acid (CPDS), another specific thiol reagent. A maximal binding of 4 mol of [14C]CPDS/mol of enzyme is obtained, concomitant to a 25% inhibition. Sequential modification of the enzyme by CPDS and [14C]NEM leads to the same final deep inactivation as that obtained with [14C]NEM alone. One out of the two thiols of the gamma-subunit is no longer accessible to [14C]NEM after CPDS treatment. When incubated at pH 6.8 with [3H]ATP in the presence of Mg2+, F1-ATPase is able to bind 3, largely exchangeable, mol of nucleotide/mol of enzyme. Modification of the three essential thiols by NEM dramatically decreases the binding of 3H-nucleotide down to about 1 mol/mol of enzyme. Partial modification modifies the cooperative properties, the enzyme being no longer sensitive to anion activation.     

Effect of ligands on the reactivity of essential sulfhydryls in brain hexokinase. Possible interaction between substrate binding sites.

Inactivation of bovine brain mitochondrial hexokinase by 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), a sulfhydryl specific reagent, has been investigated. The study shows that the inactivation of the enzyme by DTNB proceeds by way of prior binding of the reagent to the enzyme and involves the reaction of 1 mol of DTNB with a mol of enzyme. At stoichiometric levels of DTNB, the inactivation of the enzyme is accompanied by the formation of a disulfide bond. But it is not clear whether the disulfide bond or the mixed disulfide intermediate formed prior to it causes inactivation. On the basis of considerable protection afforded by glucose against this inactivation it is tentatively concluded that the sulfhydryl residues involved in this inactivation are at the glucose binding site of the enzyme, although other possibilities are not ruled out. An analysis of effects of various substrates and inhibitors on the kinetics of inactivation and sulfhydryl modification by DTNB has led to the proposal that the binding of substrates to the enzyme is interdependent and that glucose and glucose 6-phosphate produce slow conformational changes in the enzyme. Protective effects by ligands have been employed to calculate their dissociation constant with respect to the enzyme. The data also indicate that glucose 6-phosphate and inorganic phosphate share the same locus on the enzyme as the gamma phosphate of ATP and that nucleotides ATP and ADP bind to the enzyme in the absence of Mg2+.     

Modification of NAD-dependent isocitrate dehydrogenase by the 2',3'-dialdehyde derivatives of NAD, NADH, NADP, and NADPH

The 2',3'-dialdehyde nicotinamide ribose derivatives of NAD (oNAD) and NADH (oNADH) have been prepared enzymatically from the corresponding 2',3'-dialdehyde analogs of NADP and NADPH. Pig heart NAD-dependent isocitrate dehydrogenase requires NAD as coenzyme but binds NADPH, as well as NADH, ADP, and ATP, at regulatory sites. Incubation of 1-3 mM oNAD or oNADH with this isocitrate dehydrogenase causes a time-dependent decrease in activity to a limiting value 40% that of the initial enzyme, suggesting that reaction does not occur at the catalytic coenzyme site. Upon varying the concentration of oNAD or oNADH from 0.2 to 3 mM, the inactivation rate constants increase in a nonlinear manner, consistent with reversible binding of oNAD and oNADH to the enzyme prior to covalent reaction. Inactivation is accompanied by incorporation of radioactive reagent with extrapolation to 0.54 mol [14C]oNAD or 0.45 mol [14C]oNADH/mol average enzyme subunit (or about 2 mol reagent/mol enzyme tetramer) when the enzyme is maximally inactivated; this value corresponds to the number of reversible binding sites for each of the natural ligands of isocitrate dehydrogenase. The protection against oNAD or oNADH inactivation by NADH, NADPH, and ADP (but not by isocitrate, NAD, or NADP) indicates that reaction occurs in the region of a nucleotide regulatory site. In contrast to the effects of oNAD and oNADH, oNADP and oNADPH cause total inactivation of the NAD-dependent isocitrate dehydrogenase, concomitant with incorporation, respectively, of about 3.5 mol [14C]oNADP or 1.3 mol [14C]oNADPH/mol average subunit. Reaction rates exhibit a linear dependence on [oNADP] or [oNADPH] and protection by natural ligands against inactivation is not striking. These results imply that oNADP and oNADPH are acting in this case as general chemical modifiers and indicate the importance of the free adenosine 2'-OH of oNAD and oNADH for specific labeling of the NAD-dependent isocitrate dehydrogenase. The new availability of 2',3'-dialdehyde nicotinamide ribose derivatives of NAD, NADH, NADP, and NADPH may allow selection of the appropriate reactive coenzyme analog for affinity labeling of a variety of dehydrogenases.     

Apparent suicidal inactivation of DNA polymerase by adenosine 2',3'-riboepoxide 5'-triphosphate.

Adenosine 2',3'-riboepoxide 5'-triphosphate (epoxyATP) has been found to be a suicidal inactivator of DNA polymerase I from Escherichia coli by the following criteria. Inactivation is complete, is first order in enzyme activity, and shows saturation kinetics with an apparent KD of 30 +/- 10 micron for epoxy ATP. This KD is comparable to the KM of the substrate dATP. The t1/2 for inactivation is 1.3 min. Inactivation requires Mg2+ and the complementary template. The enzyme is protected by dATP but not by an excess of template. Gel filtration of the reaction mixture after inactivation with [3H]epoxy ATP results in the comigration of E. coli DNA polymerase I, the tritium-labeled inactivator, and the DNA template. The stoichiometry of binding approaches 1 mol of [3H]epoxy nucleotide per mol of inactivated enzyme. These results are consistent with the hypothesis that epoxy ATP initially serves as a substrate for the polymerase reaction, elongating the DNA chain by a nucleotidyl unit, and subsequently alkylates an essential base at the primer terminus binding site of the enzyme. Epoxy ATP also inactivates human and viral DNA polymerases but not E. coli RNA polymerase or rabbit muscle pyruvate kinase. Hence epoxy ATP may be a specific suicide reagent for DNA polymerases.     

Inactivation of NAD-dependent isocitrate dehydrogenase by affinity labeling of the allosteric ADP site by 2-(4-bromo-2,3-dioxobutylthio)adenosine 5'-diphosphate

A new reactive ADP analogue has been synthesized: 2-(4-bromo-2,3-dioxobutylthio)adenosine 5'-diphosphate (2-BDB-TADP). Reaction of ADP with m-chloroperoxybenzoic acid gave ADP 1-oxide, which was treated with NaOH, followed by reaction with carbon disulfide to yield 2-thioadenosine 5'-diphosphate. The final product was synthesized by condensation of 2-thioadenosine 5'-diphosphate with 1,4-dibromobutanedione. Reaction of pig heart NAD-specific isocitrate dehydrogenase with this nucleotide analogue (0.4 mM) causes a time-dependent loss of activity to a limiting value of 75% inactivation. The rate constant for inactivation exhibits a nonlinear dependence on the concentration of 2-BDB-TADP, with kmax = 0.021 min-1 and KI = 0.067 mM. Complete protection against inactivation by 0.2 mM 2-BDB-TADP is provided by ADP + Mn2+, but not by Mn2+ alone, isocitrate, alpha-ketoglutarate, or NAD. Incorporation of 2-BDB-TADP is proportional to the extent of inactivation, reaching 1 mol of reagent/mol of enzyme subunit when the enzyme is maximally inactivated. However, when inactivation is totally prevented by incubation with 2-BDB-TADP in the presence of ADP and Mn2+, 0.5 mol of reagent/mol of subunit is still incorporated, suggesting that inactivation may be attributed to 0.5 mol of reagent/mol of average subunit. In the native enzyme, the Km for total isocitrate is 1.8 mM and is decreased 6-fold to 0.3 mM in the presence of 1 mM ADP, whereas in the modified enzyme, with 25% residual activity, the Km for total isocitrate is about the same in the absence (2.0 mM) or presence (1.8 mM) of ADP. These results indicate that 2-BDB-TADP acts as an affinity label of the ADP allosteric site of NAD-dependent isocitrate dehydrogenase.     

Photoaffinity labeling of soluble chloroplast adenosine 5′-triphosphatase with 3′-O-(4-benzoyl)benzoyl ADP

3′-O-(4-benzoyl)benzoyl ADP (BzADP) acts as a reversible inhibitor of the chloroplast coupling factor 1 ATPase (CF₁) when incubated with the enzyme in the dark. The V_max of ATP hydrolysis is decreased and the kinetics of the reaction are altered from noncooperative to cooperative with respect to ATP. Photoactivation of the benzophenone group in BzADP by irradiation with ultraviolet light (366 nm) results in the covalent binding of BzADP to the enzyme and inactivation of its enzymic activity. Polyacrylamide gel electrophoresis of CF₁-ATPase in the presence of sodium dodecyl sulfate shows that the analog is bound primarily to the enzyme's β subunit. Complete inactivation of the activated CF₁-ATPase occurs upon covalent binding of 2.45 mol BzADP/mol CF₁. Binding of BzADP and inactivation of the ATPase are prevented if ADP, but not ATP, is present during the photoactivation step. The presence of Ca²⁺ during irradiation enhances the rate of BzADP covalent binding as well as the rate of inactivation of the enzyme.     

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

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

Saccharomyces cerevisiae phosphoenolpyruvate carboxykinase: physicochemical characteristics of the nucleotide binding site, as deduced from fluorescent spectroscopy measurements

Saccharomyces cerevisiae phosphoenolpyruvate carboxykinase [ATP:oxaloacetate carboxy-lyase (transphosphorylating), EC 4.1.1.49] is inactivated by the fluorescent sulfhydryl reagent N-(iodoacetyl-N'-(5-sulfo-1-naphthyl)ethylenediamine (1,5-IAEDANS). The inactivation reaction follows pseudo-first-order kinetics with respect to active enzyme to less than 10% remaining enzyme activity, with a second-order inactivation rate constant of 2.6 min-1 mM-1 at pH 7.5 and 30 degrees C. A stoichiometry of 1.05 mol of reagent incorporated per mole of enzyme subunit was found for the completely inactivated enzyme. Almost complete protection of the enzyme activity and of dansyl label incorporation are afforded by MnADP or MnATP, thus suggesting that 1,5-IAEDANS interacts with an enzyme sulfhydryl group at the nucleotide binding site. The fluorescence decay of the AEDANS attached to the protein shows a single-exponential behavior with a lifetime of 18 ns. A comparison of the fluorescence band position and the fluorescence decay with those of the adduct AEDANS-acetylcysteine indicates a reduced polarity for the microenvironment of the substrate binding site. The quenching of the AEDANS moiety in the protein can be described in terms of a collisional and a static component. The rate constant for the collisional component is much lower than that obtained for the adduct in a medium of reduced polarity. These last results indicate that the AEDANS moiety is considerably shielded from the solvent when it is covalently attached to PEPCK.     

Heart phosphofructokinase. Allosteric kinetics following affinity labeling modification of the enzyme by 8-[m-(m-fluorosulfonylbenzamido) benzylthio] adenine.

An adenine analog 8-[m-(m-fluorosulfonylbenzamido)benzylthio]adenine (FSB-adenine) reacts covalently with sheep heart phosphofructokinase. Under conditions optimal for allosteric kinetics the modified enzyme is less sensitive to inhibition by ATP and insensitive to activation by AMP, cyclic AMP, and ADP. The concentration of fructose-6-P necessary for half-maximal activity is markedly decreased, while the cooperativity to the same substrate is not changed under the same conditions. The modified enzyme is more stable at pH 6.5 when compared with the native enzyme. Changes in the allosteric kinetics of the enzyme are proportional to the extent of modification reaching maximal effect when 3.2 mol of the reagent were bound/mol of tetrameric enzyme. Affinity labeling of the enzyme by the adenine derivative does not affect significantly the catalytic site. This is evidenced by the demonstration that under assay conditions optimal for Michaelian kinetics neither the Km for ATP nor for fructose-6-P is significantly changed following chemical modification. Maximal activity of the modified enzyme was 60% of the native enzyme. ADP gives the best protection, while AMP gives less protection against modification by the reagent. ATP slows the rate of the reaction and causes a slight decrease in maximum binding of the reagent to the enzyme. Modification of the enzyme caused a marked reduction of AMP and ADP binding. The evidence indicates that the modified site is a nucleotide mono- and diphosphate activation site.     

Does 2-hydroxy-5-nitrobenzyl bromide react with the epsilon-subunit of the mitochondrial F1-ATPase?

The incubation of bovine mitochondrial F1-ATPase with 2-hydroxy-5-nitrobenzyl bromide (HNB), a selective reagent toward tryptophan residues in proteins, produced a concentration dependent inactivation of the enzyme and the covalent binding of 0.88 mol reagent/mol F1. Although HNB is highly specific for tryptophan it has also some reactivity toward cysteine, then a pre-treatment of F1 with several sulphydryl reagents has been performed to make the site of reaction clearer. This pre-treatment had neither effects in the binding stoichiometry nor in the extent of catalytic inhibition, suggesting that readly accessible thiol groups are not involved in the reaction with HNB. Since the only tryptophan bearing polypeptide of the bovine mitochondrial F1-ATPase complex is its smallest subunit, subunit-epsilon, this is the most probable candidate for HNB reaction. Therefore it may be inferred that the intactness and/or the correct conformation of this subunit could be important factor(s) for the multisite ATP hydrolytic activity of the enzyme.     

Reactive sulfhydryl groups in Saccharomyces cerevisiae phosphoenolpyruvate carboxykinase

Saccharomyces cerevisiae phosphoenolpyruvate carboxykinase (ATP:oxaloacetate carboxy-lyase (transphosphorylating), EC 4.1.1.49) is inactivated by several thiol- and vicinal dithiol-specific reagents. Titration experiments of the enzyme with 5,5'-dithiobis(2-nitrobenzoate) (DTNB) show the presence of reactive monothiol and vicinal dithiol groups, whose modifications lead to enzyme inactivation. The enzyme is also inactivated by N-(1-pyrenyl)iodoacetamide (PyrIAM), with a binding stoichiometry of approx. 2 mol per mol of enzyme subunit. A high level of pyrene excimer fluorescence is detected on the labeled enzyme, thus implying the reaction of the reagent with two spatially close sulfhydryl groups in the protein. The carboxykinase is not completely inactivated by different vicinal dithiol-specific reagents, thus implying a catalytically non-essential character for these groups. From substrate protection experiments of the enzyme inactivation by DTNB, PyrIAM and vicinal dithiol-specific reagents, it is concluded that the loss of enzyme activity is caused by the modification of both thiol and vicinal dithiol groups in the substrate binding region.     

Inactivation of the amidotransferase activity of carbamoyl phosphate synthetase by the antibiotic acivicin.

Carbamoyl phosphate synthetase (CPS) from Escherichia coli catalyzes the formation of carbamoyl phosphate from 2 mol of ATP, bicarbonate, and glutamine. CPS was inactivated by the glutamine analog, acivicin. In the presence of ATP and bicarbonate the second-order rate constant for the inactivation of the glutamine-dependent activities was 4.0 x 10(4) m(-1) s(-1). In the absence of ATP and bicarbonate the second-order rate constant for inactivation of CPS was reduced by a factor of 200. The enzyme was protected against inactivation by the inclusion of glutamine in the reaction mixture. The ammonia-dependent activities were unaffected by the incubation of CPS with acivicin. These results are consistent with the covalent labeling of the glutamine-binding site located within the small amidotransferase subunit. The binding of ATP and bicarbonate to the large subunit of CPS must also induce a conformational change within the amidotransferase domain of the small subunit that enhances the nucleophilic character of the thiol group required for glutamine hydrolysis. The acivicin-inhibited enzyme was crystallized, and the three-dimensional structure was determined by x-ray diffraction techniques. The thiol group of Cys-269 was covalently attached to the dihydroisoxazole ring of acivicin with the displacement of a chloride ion.