Structural mapping of rabbit muscle phosphofructokinase. Distance between the adenosine cyclic 3',5'-monophosphate binding site and reactive sulfhydryl group

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: 80%

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

Tomich¹,

Martí²,

Colman³

1981

“…Radioactive 4-(iodoacetamido)salicylic acid was prepared from iodo[l-14C]acetic acid (New England Nuclear Corp.) as described by Bacon et al (1981). 5 '-[p-(Fluorosulfonyl)benzoyl]-2-aza-l^-ethenoadenosine was synthesized by the procedure of Craig & Hammes (1980); 2-aza-, 6ethenoadenosine was from Molecular Probes, and p-(fluorosulfonyl)benzoyl chloride was from Aldrich Chemical Co. TNP-ADP and 2-aza-l.M-ethenoadenosine S'-triphosphate were also purchased from Molecular Probes. [8-14C] ADP was purchased from New England Nuclear Corp. All coenzymes and purine nucleotides, as well as EDTA and Tris base, were purchased from Sigma Chemical Co. Analytically pure samples of 0-[(4-carboxyphenyl)sulfonyl]tyrosine and Are-[(4carboxyphenyl)sulfonyl]lysine prepared by Saradambal et al (1981) were made available to us.…”

Section: Methodsmentioning

confidence: 99%

“…The fluorescent label 5Z-FSB<A previously used to modify a GTP site does not exhibit appropriate spectral overlap with ISA. The fluorescent nucleotide analogue 5'-[p-(fluorosulfonyl)benzoyl] -2-aza-1 .A^-ethenoadenosine (5'-FSBaeA) is structurally similar to 5'-FSBeA but possesses different spectral properties (Xabs = 356 nm, Xemiss = 490 nm) (Craig & Hammes, 1980) and qualifies as an acceptor for the salicylate donor fluorescence. The reaction of S'-FSBaeA with glutamate dehydrogenase is described in this paper, and the distances between the catalytic and regulatory sites are measured by fluorescence energy transfer.…”

mentioning

confidence: 99%

Distance relationships between the catalytic site labeled with 4-(iodoacetamido)salicylic acid and regulatory sites of glutamate dehydrogenase

Jacobson¹,

Colman²

1984

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.

“…Examples of proteins for which such distance measurements have been made are chloroplast coupling factor 1 (Cantley & Hammes, 1975), pyruvate dehydrogenase complex (Angelides & Hammes, 1979), galactosyltransferase (O'Keeffe et al, 1980), glutamate dehydrogenase (Jacobson & Colman, 1984), phosphofructokinase (Craig & Hammes, 1980), G-actin (Miki & Wahl, 1985), and glutamine synthetase (Maurizi et al, 1986). The coenzyme analogue thionicotinamide adenine dinucleotide phosphate (TNADP+) is a substrate for isocitrate dehydrogenase (Stein et al, 1963).…”

mentioning

confidence: 99%

Distances among coenzyme and metal sites of NADP+-dependent isocitrate dehydrogenase using resonance energy transfer

Bailey¹,

Colman²

1987

When the substrate isocitrate-Mn2+ is present, the fluorescent 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 pig heart NADP+-specific isocitrate dehydrogenase at the coenzyme binding site on one subunit of the dimeric enzyme [Bailey, J. M., & Colman, R. F. (1985) Biochemistry 24, 5367-5377]. The modified enzyme, which retains partial activity, binds 1 mol of NADPH or 1 mol of the coenzyme analogue, reduced thionicotinamide adenine dinucleotide phosphate (TNADPH), per dimer. TNADPH quenches the fluorescence of enzyme-bound 2-BDB-T epsilon A-2',5'-DP with an efficiency of energy transfer of 9.8%. From this value and the spectral properties of the donor and acceptor chromophores, a distance of 32 A was calculated as the average distance between coenzyme sites on the two subunits. Isocitrate dehydrogenase activity requires a divalent metal ion, such as Mn2+, Co2+, or Ni2+. Co2+ and Ni2+ have absorption spectra that overlap the emission spectra of enzyme-bound 2-BDB-T epsilon A-2',5'-DP. In the presence of isocitrate, each of these two metal ions quenches the fluorescence of the enzyme-bound reagent with an efficiency of energy transfer of 28-29%. From this value and the spectral characteristics of the energy donor and acceptors, an average distance of 8.0 A was estimated between the metal-isocitrate site and the labeled coenzyme site. These distances have provided constraints in formulating a model of the spatial arrangement of active-site ligands on isocitrate dehydrogenase.