Browsing by Author "DEOVANDO, FS"

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    POSSIBLE INVOLVEMENT OF CYSTEINYL, LYSYL AND ARGINYL GROUPS IN THE ACTIVE-CENTER OF PIG-LIVER PHOSPHOMEVALONATE KINASE
    (1981) BAZAES, S; BEYTIA, E; JABALQUINTO, AM; DEOVANDO, FS; GOMEZ, I; EYZAGUIRRE, J
    Pig liver phosphomevalonate kinase has a MW of about 22,000, its structure is monomeric and it shows only 1 cysteine residue/molecule. The presence of cysteinyl and lysyl residues at the active site was studied by chemical modification. Preliminary work was also performed on the involvement of arginyl residues. Modification of cysteinyl residues was attempted using 5,5''dithiobis (2-notrobenzoate) (DTNB). Pyridoxal phosphate (PLP) was utilized as reagent for lysyl residues, and arginyl residues were modified with butanedione. Inactivation of the enzyme by DTNB is complete but may be reversed by 2-mercaptoethanol or dithiothreitol. Kinetic analysis shows that 1 mol of reactive SH group/mol of enzyme is responsible for catalytic activity. The substrate phosphomevalonate, but not Mg-ATP, protects against inactivation. Since no change in Km or kcat is observed on partial inactivation, this is interpreted as indication that the modified molecule is totally inactive. PLP inactivates the enzyme, but even at high concentrations total inactivation was not observed. Addition of lysine or dialysis reactivates the modified enzyme, but this does not occur after the addition of sodium borohydride. Pyridoxal and pyridoxamine 5''-phosphate are weaker inhibitors than PLP. As with DTNB, phosphomevalonate but not MgADP protects against inactivation. The partially modified enzyme shows a lower Km for phosphomevalonate than the native enzyme. Kinetic data suggest that 1 PLP molecule reacts/enzyme active center, while spectrophotometric titration shows a lower value. pK values 7.2 (at 24.degree. C) and 7.5 (at 31.degree. C) are obtained for the modified group, in agreement with values described for reactive lysines. Inactivation by butanedione can be protected by phosphomevalonate, but only to a smaller extent by Mg-ATP. The cysteinyl residue of the enzyme plays an essential role in the catalytic mechanism, while the lysine residue modified may not be essential for activity. Both residues, and possibly an arginyl group, may be located at or near the phosphomevalonate binding site of the enzyme.