Farnesylation of nonpeptidic thiol compounds by protein farnesyltransferase.
Protein farnesyltransferase catalyzes the modification of protein substrates containing specific carboxyl-terminal Ca(1)a(2)X motifs with a 15-carbon farnesyl group. The thioether linkage is formed between the cysteine of the Ca(1)a(2)X motif and C1 of the farnesyl group. Protein substrate specificity is essential to the function of the enzyme and has been exploited to find enzyme-specific inhibitors for antitumor therapies. In this work, we investigate the thiol substrate specificity of protein farnesyltransferase by demonstrating that a variety of nonpeptidic thiol compounds, including glutathione and dithiothreitol, are substrates. However, the binding energy of these thiols is decreased 4-6 kcal/mol compared to a peptide derived from the carboxyl terminus of H-Ras. Furthermore, for these thiol substrates, both the farnesylation rate constant and the apparent magnesium affinity decrease significantly. Surprisingly, no correlation is observed between the pH-independent log(k(max)) and the thiol pK(a); model nucleophilic reactions of thiols display a Brønsted correlation of approximately 0.4. These data demonstrate that zinc-sulfur coordination is a primary criterion for classification as a FTase substrate, but other interactions between the peptide and the FTase.isoprenoid complex provide significant enhancement of binding and catalysis. Finally, these results suggest that the mechanism of FTase provides in vivo selectivity for the farnesylation of protein substrates even in the presence of high concentrations of intracellular thiols.
Hightower
K E
KE
Department of Biochemistry, Duke University Medical Center, Durham, North Carolina 27710, USA.
Casey
P J
PJ
Fierke
C A
CA
D019883:Alkyl and Aryl Transferases / Q000737:chemistry / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D002384:Catalysis; D004229:Dithiothreitol / Q000737:chemistry; D005978:Glutathione / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008274:Magnesium / Q000737:chemistry; D010455:Peptides / Q000378:metabolism; D017368:Protein Prenylation; D051381:Rats; D019163:Reducing Agents / Q000737:chemistry; D013379:Substrate Specificity; D013438:Sulfhydryl Compounds / Q000737:chemistry / Q000378:metabolism*
D010455:Peptides; D019163:Reducing Agents; D013438:Sulfhydryl Compounds; D019883:Alkyl and Aryl Transferases; C064911:p21(ras) farnesyl-protein transferase; D005978:Glutathione; D008274:Magnesium; D004229:Dithiothreitol