CHEMICAL MODIFICATION OF ESSENTIAL ARGININES IN PIG-LIVER PHOSPHOMEVALONATE KINASE
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Date
1982
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Abstract
Phosphomevalonate [MVAP] kinase, an enzyme of the polyisoprenoid biosynthesis pathway, catalyzes the transfer of phosphate from ATP to MVAP, with the formation of pyrophosphomevalonate and ADP. The pig liver enzyme, a monomer of MW 22,000, possesses 1 cysteinyl residue, which is essential for catalysis. By means of chemical modification of a partially purified preparation, the participation of arginine residues in the enzyme active site was studied. Butanedione and phenylglyoxal were chosen as group-specific reagents. The kinetics of inactivation by both reagents is rather complex, suggesting that several arginine residues, directly or indirectly related to the active site, are being modified. Both substrates, MVAP and Mg-ATP, protect against inactivation but to a different extent, depending on the modifier used. With butanedione, almost total protection is achieved with Mg-ATP. Better protection with MVAP is observed for the modification with phenylglyoxal, but with this reagent, Mg-ATP protects strongly only at very high concentrations. A reversible inactivation mechanism is followed with butanedione, while for phenylglyoxal this mechanism appears to be irreversible, in agreement with findings by other authors. MVAP kinase probably presents 2 or more arginyl residues in or near its active site, one of them being involved in the binding of Mg-ATP, and at least another located in the neighborhood of the MVAP binding site. A more precise determination of the number of essential residues requires its measurement by chemical methods, utilizing a homogeneous enzyme preparation.