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

Abstract

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.

Levels of inflammation and oxidative stress, and a role for taurine in dystropathology of the Golden Retriever Muscular Dystrophy dog model for Duchenne Muscular Dystrophy

“…Previous research shows that thiol oxidation of these proteins can directly effect their functions [88], [89], [90], [91], [92], [93]. While the consequences of high thiol oxidation of these diverse proteins in GRMD muscle are yet to be fully understood and appreciated, functional changes in these proteins and downstream cellular consequences may well contribute to severity of dystropathology in GRMD dogs.…”

Evolution of Negative Cooperativity in Glutathione Transferase Enabled Preservation of Enzyme Function

“…Alternatively, the binding of the inhibitor to the first subunit may give the second subunit lower affinity for the same compound. In this case the adjacent subunit retains its original activity even in the presence of an excess of inhibitor (43). In this complex scenario of different and sometimes opposite effects triggered by negative cooperativity mechanisms, a much debated question is the identification of specific physiological functions.…”

Requirement of two specific tyrosine residues for the catalytic activity of Bcr serine/threonine kinase

“…What precise roles these two tyrosine play in the catalytic mechanism of the Bcr kinase is not known. Some hints as to the role of these tyrosines can be gained from the knowledge of the catalytic mechanism of pyruvate kinase (Annamalai and Colman, 1981;Tomich et al, 1981). Their studies indicate that a critical tyrosine residue and two cysteine pairs play an important role in pyruvate kinase activity (Annamalai and Colman, 1981).…”

“…A second pair of cysteines is present at residues 388 and 395. Similar cysteine pairs are thought to be involved in other novel kinases such as pyruvate kinase (Tomich et al, 1981).…”