Essential arginine residues in isoprenylcysteine protein carboxyl methyltransferase.

We used specific amino acid modifying reagents to characterize the isoprenylcysteine carboxyl methyltransferase in kidney membranes. The enzyme was inactivated by reagents specific for arginine, histidine, cysteine, and tryptophan residues. Protection by the product and inhibitor S-adenosyl-L-homocysteine was observed for arginine modification by phenylglyoxal and tryptophan modification by N-bromosuccinimide. We focused on modification by phenylglyoxal, a highly specific modifier of arginine residues. The inactivation of methyltransferase by phenylglyoxal follows pseudo-first-order kinetics and the order of the reaction, n, with respect to phenylglyoxal was 1.2. The inactivation increased with the alkalinity of the preincubation medium and was maximal at pH 10. Kinetic analysis showed that the K(m) for S-adenosyl-L-methionine is not significantly affected by treatment with phenylglyoxal but that the Vmax is reduced p-Hydroxyphenylglyoxal, a more hydrophilic derivative of phenylglyoxal, was a less potent inactivator of methyltransferase than phenylglyoxal, suggesting that arginine residues modified are in a hydrophobic environment. The methyltransferase is protected from phenylglyoxal modification by S-adenosyl-L-homocysteine but not S-adenosyl-L-methionine, sinefungin, N-acetyl-S-farnesyl-L-cysteine, or farnesylthioacetate. The arginine residue modified may thus be located either at the active site or at another additional binding site for S-adenosyl-L-homocysteine. These results indicate that arginine residues are essential for the enzymatic activity of isoprenylcysteine carboxyl methyltransferase.