Kinetic mechanism of isoprenylated protein methyltransferase.
The kinetic mechanism of the rod outer segment (ROS) isoprenylated protein methyltransferase was investigated. This S-adenosyl-L-methionine (AdoMet)-linked enzyme transfers methyl groups to carboxyl-terminal isoprenylated cysteine residues of proteins, generating methyl esters. The enzyme also processes simple substrates such as N-acetyl-S-farnesyl-L-cysteine (L-AFC). Initial studies showed that a ping-pong Bi Bi mechanism could be eliminated. In a ping-pong Bi Bi mechanism plots of 1/v versus 1/[substrate A] at different fixed substrate B concentrations are expected to yield a family of parallel lines whose slopes equal Km/Vmax. In fact, converging curves were found, which suggested a sequential mechanism. Dead-end inhibitors were used in order to further investigate the kinetic mechanism. S-Farnesylthioacetic acid is shown to be a dead-end competitive inhibitor with respect to the prenylated substrate L-AFC. On the other hand, S-farnesylthioacetic acid proved to be uncompetitive with respect to AdoMet, suggesting an ordered mechanism with AdoMet binding first. Further evidence for this mechanism came from product inhibition studies using the methyl ester of L-AFC (L-AFCMe) and S-adenosyl-L-homocysteine (AdoHcy). Since AdoMet binds first to the enzyme, one of the products (L-AFCMe or AdoHcy) should be a competitive inhibitor with respect to it. It could be shown that AdoHcy is a competitive inhibitor with respect to AdoMet, but L-AFCMe is a mixed-type inhibitor both with respect to AdoMet and to L-AFC. Therefore, AdoHcy combines with the same enzyme form as does AdoMet, and must be released from the enzyme last. Moreover, L-AFC and L-AFCMe must bind to different forms of the enzyme.
Shi
Y Q
YQ
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115.
Rando
R R
RR
D000818:Animals; D001667:Binding, Competitive; D002417:Cattle; D008962:Models, Theoretical; D011496:Protein Methyltransferases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D012374:Rod Cell Outer Segment / Q000201:enzymology*; D012436:S-Adenosylmethionine / Q000378:metabolism; D013379:Substrate Specificity
D012436:S-Adenosylmethionine; D011496:Protein Methyltransferases; C056501:protein-S-isoprenylcysteine O-methyltransferase