DIFFERENTIAL PRENYL PYROPHOSPHATE BINDING TO MAMMALIAN PROTEIN GERANYLGERANYLTRANSFERASE-I AND PROTEIN FARNESYLTRANSFERASE AND ITS CONSEQUENCE ON THE SPECIFICITY OF PROTEIN PRENYLATION

Protein geranylgeranyltransferase-I (PGGT-I) and protein farnesyltrans ferase (PFT) attach geranylgeranyl and farnesyl groups, respectively, to the C termini of eukaryotic cell proteins, In vitro, PGGT-I and PFT can transfer both geranylgeranyl and farnesyl groups from geranylgera nyl pyrophosphate (GGPP) and farnesyl pyrophosphate (FPP) to their pro tein or peptide prenyl acceptor substrates. In the present study it is shown that PGGT-I binds GGPP 330-fold tighter than FPP and that PFT b inds FPP 15-fold tighter than GGPP, Therefore, in vivo, where both GGP P and FPP compete for the binding to prenyltransferases, PGGT-I and PF T will likely be bound predominantly to GGPP and FPP, respectively. Pr evious studies have shown that K-Ras4B and the Ras-related GTPase TC21 are substrates for both PGGT-I and PFT in vitro, It is shown that TC2 1 can compete with the C-terminal peptide of the gamma subunit of hete rotrimeric G proteins and with the C-terminal peptide of lamin B for g eranylgeranylation by PGGT-I and for farnesylation by PFT, respectivel y, K-Ras4B competes in both cases but is almost exclusively farnesylat ed by PFT in the presence of the lamin B peptide competitor. Rapid and single turnover kinetic studies indicate that the rate constant for t he PGGT-I-catalyzed geranylgeranyl transfer step of the reaction cycle is 14-fold larger than the steady-state turnover number, which indica tes that the rate of the overall reaction is limited by a step subsequ ent to prenyl transfer such as release of products from the enzyme, PG GT-I-catalyzed farnesylation is 37-fold slower than geranylgeranylatio n and is limited by the farnesyl transfer step, These results together with earlier studies provide a paradigm for the substrate specificity of PGGT-I and PFT and provide information that is critical for the de sign of prenyltransferase inhibitors as anti-cancer agents.