SUBSTRATE-BINDING IS REQUIRED FOR RELEASE OF PRODUCT FROM MAMMALIAN PROTEIN FARNESYLTRANSFERASE

Protein farnesyltransferase (FTase) catalyzes the modification by a fa rnesyl lipid of Pas and several other key proteins involved in cellula r regulation. Previous studies on this important enzyme have indicated that product dissociation is the rate-limiting step in catalysis. A d etailed examination of this has now been performed, and the results pr ovide surprising insights into the mechanism of the enzyme. Examinatio n of the binding of a farnesylated peptide product to free enzyme reve aled a binding affinity of similar to 1 mu M. However, analysis of the product release step under single turnover conditions led to the surp rising observation that the peptide product did not dissociate from th e enzyme unless additional substrate was provided. Once additional sub strate was provided, the enzyme released the farnesylated peptide prod uct with rates comparable with that of overall catalysis by FTase. Add itionally, stable FTase-farnesylated product complexes were formed usi ng Ras proteins as substrates, and these complexes also require additi onal substrate for product release. These data have major implications in both our understanding of overall mechanism of this enzyme and in design of inhibitors against this therapeutic target.