In the collaboration, Beese has used X-ray crystallography to develop a detailed structure of the FTase enzyme. Fierke has used biochemical and biophysical techniques to understand the detailed mechanism of the enzyme, and Casey has performed kinetic and structure-function studies.
In Casey's studies, he and graduate student Ruth Fu have produced versions of FTase with selectively altered amino acids in the active site to test hypotheses about which amino acid residues are important in binding the farnesyl and Ras molecules and in carrying out the catalytic reaction to attach the two.
"We now have a very good idea of exactly how the chemistry of the reaction occurs," said Casey, "information that has eluded us for several years."
This chemical information comes primarily from the latest studies in Fierke's laboratory showing clearly that zinc plays an important role in catalyzing the reaction, Casey said. The studies by Fierke and graduate student Chih-chin Huang show that the zinc in the FTase enzyme interacts catalytically with the sulfur in the Ras protein in the process of joining the reactive farnesyl di-phosphate molecule with the Ras protein. In experiments in which they substituted for zinc in the enzyme other metals that more tightly bind sulfur, the biochemists were able to "freeze" the reaction to better understand the mechanism. The biochemists also tested the effects of altered farnesyl molecules -- with the two kinds of experiments revealing that the FTase reaction is a "carbo-cation nucleophile mixed mechanism."
Fierke and postdoctoral fellow Kendra Hightower -- along with
postdoctoral fellow Becky Spense in the Casey lab -- are exploring how both
FTase and a related enzyme called geranylgeranyl transferase bind different
substrate molecules. This study is an attempt to understand differences between
these two enzymes that might aid developmen
Contact: Dennis Meredith