Affinity labeling of AMP-ADP sites in heart phosphofructokinase by 5′-p-fluorosulfonylbenzoyl adenosine

Reaction of rabbit muscle pyruvate kinase with the affinity label 5'-[p-(fluorosulfonyl) benzoyl] guanosine (5'-FSBG), at pH 7.65 and 7.93, leads to a loss in enzyme activity. The inactivation is characterized by a biphasic kinetic profile, with the initial phase accounting for approximately 55% of the reduction in enzymatic activity. For both the rapid and slow phases, at pH 7.93, the inactivation rate constants are linearly proportional to the reagent concentration (from 0.48 to 3.0 mM), yielding second-order rate constants of 195 min-1 M-1 and 19 min-1 m-1, respectively. The effect of ligands was tested on the two phases of inactivation. For both, a decrease in the inactivation rate was produced by Mg2+ alone, but the best protection was provided by Mg2+ plus either ADP or GDP, suggesting that the reaction occurs in the region of the metal-nucleotide binding site. Modified pyruvate kinase is completely reactivated by incubation with 20 mM dithiothreitol, indicating the involvement of cysteine in the inactivation, indicating the involvement of cysteine in the inactivation process. Reaction with [5'=3H]-5'-FSBG leads to the incorporation of up to 1.3 mol of radioactive reagent per mol of enzyme subunit; however, identical radiolabel incorporation is observed before or after dithiothreitol reactivation of modified enzyme. This result implies that the labeled amino acid residue, measured by means of incorporation, is not directly involved in the inactivation process. In contrast, inactivation was found to correlate well with the loss of two free sulfhydryl groups per enzyme subunit and the restoration of activity to correlate with the regeneration of two free sulfhydryls after treatment of modified enzyme with dithiothreitol. It is proposed that inactivation of pyruvate kinase by 5'-FSBG proceeds by formation of thiol sulfonate followed by a rapid displacement of the sulfinic acid moiety by a second cysteine to yield a disulfide. A negative cooperatively in the interaction of pyruvate kinase subunits with 5'-[p-(fluorosulfonyl)-benzoyl] guanosine might best account for the biphasic inactivation kinetics.

Section: Discussionmentioning

confidence: 81%

Modification of two essential cysteines in rabbit muscle pyruvate kinase by the guanine nucleotide analog 5'-[p-(fluorosulfonyl)benzoyl]guanosine

Tomich¹,

Martí²,

Colman³

1981

“…The reactivation by dithiothreitol of pyruvate kinase which had been inactivated by 5'-[p-(fluorosulfonyl)benzoyl]-TA^-ethenoadenosine may have important implications for studies previously reported on the modification of several enzymes by 5'-[/i-(fluorosulfonyl)benzoyl]adenosine. In a number of cases, dithiothreitol or mercaptoethanol was used to stop the reaction between 5'-FS02BzAdo and the enzyme (Pettigrew & Frieden, 1978;Mansour & Colman, 1978;Esch & Allison, 1978;Zoller & Taylor, 1979). The results of the present study indicate the need to ascertain the effect of added thiol on the activity and extent of reagent incorporation for these other modified enzymes.…”

Section: Discussionmentioning

confidence: 86%

“…-Affinity labeling using nucleotide analogues with reactive functional groups has proved to be an effective approach to exploring the nucleotide binding sites of proteins ; Hampton et al, 1977;Gulyaev et al, 1976). Among the most generally applicable of the nucleotide alkylating agents is 5'-[p-(fluorosulfonyl)benzoyl] adenosine, which labels specifically nucleotide binding sites of glutamate dehydrogenase (Pal et al, 1975), rabbit muscle and yeast pyruvate kinases Likos et al, 1980), phosphofructokinase (Mansour & Colman, 1978; Pettigrew & Frieden, 1978;Weng et al, 1980), mitochondrial F, AT-Pase1 **** (Esch & Allison, 1978;DiPietro et al, 1979), chloroplast ATPase (DeBenedetti & Jagendorf, 1979), cAMP-dependent protein kinase (Zoller & Taylor, 1979; Hixson & Krebs, 1979), malate dehydrogenase (Roy & Colman, 1979), glutamine synthetase (Foster & Kingdon, 1980), and an ADP receptor protein of platelets (Bennett et al, 1978;Mills et al, 1980). Fluorescent analogues of natural biochemical compounds as well as fluorescent labeling agents have been valuable in probing the environment of binding sites in proteins and in + From the Department of Chemistry, University of Delaware, Newark, Delaware 19711.…”

mentioning

confidence: 99%

Affinity labeling of rabbit muscle pyruvate kinase by a new fluorescent nucleotide alkylating agent, 5'-[p-(fluorosulfonyl)benzoyl]-1,N6-ethenoadenosine

Likos¹,

Colman²

1981

This paper describes the synthesis and characterization of a new fluorescent nucleotide analogue, 5'-[p-(fluorosulfonyl)benzoyl]-1 ,M-ethenoadenosine, which is capable of reacting covalently with nucleophilic groups in proteins. This nucleotide analogue, with a fluorescence emission maximum at 412 nm, functions as an active site directed irreversible inhibitor of rabbit muscle pyruvate kinase. The inactivation follows pseudo-first-order kinetics at pH 8.0. A plot of the rate constant for inactivation vs. the analogue concentration yields hyperbolic kinetics, indicative of reversible binding of the analogue prior to covalent reaction. Significant protection is afforded by phosphoenolpyruvate, while MgATP, MgADP, and Mg alone decrease the rate of inactivation but not to the same extent as does phosphoenolpyruvate. The metal-free nucleotides ADP or ATP as well as pyruvate have no effect on the rate of inactivation. The incorporation of

“…The activating adenine nucleotide binding site can be specifically labeled by 5'-FS02BzAdo, and the covalent attachment of the affinity label to this site results in a relief of inhibition by ATP similar to that produced by the noncovalent binding of cAMP to the site (Pettigrew & Frieden, 1978; Ogilvie, 1983). The activating adenine nucleotide binding site of sheep heart PFK also undergoes specific labeling by 5'-FS02BzAdo (Mansour & Colman, 1978), and the site of modification is a lysine residue (Weng et al, 1980).…”

mentioning

confidence: 99%

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

Ogilvie¹

1985

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.