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Transition state analysis of the chemical and enzymatic prenylation reactions.

Recent Research from the group of Professor Mark Distefano.

Protein prenylation involves the attachment of C15 (farnesyl) or C20 (geranylgeranyl) groups to proteins and is catalyzed by a class of enzymes known as prenyltransferases. The observation that inhibition of Ras farnesylation arrests the growth of tumor cells has been the motivating factor in developing inhibitors of prenyltransferases that can serve as anticancer drugs; currently several candidates are in Phase 3 clinical trials. We are interested in using kinetic isotope effect (KIE) measurements to determine the transition state (TS) structure for the enzyme catalyzed reaction since knowledge of the TS structure may allow the selectivity and affinity of inhibitors of these enzymes to be improved. Recently, graduates students Stepan Lenevich and Ayako Hosakawa, and Postdoctoral Fellow Dr. Juhua Xu in the research group of Professor Mark Distefano measured a primary 13C KIE and a secondary 2H KIE via mass spectrometry. In collaboration with Professor Chris Cramer, a TS structure for the farnesyltransferase enzyme-catalyzed reaction was computed; a density functional level of electronic structure theory using the m PW1N functional in combination with the 6-31+G(d) basis set was employed for those calculations. The results indicate that the enzyme effects catalysis via an “exploded” TS structure with an extended C-S bond. This is the first example of a transition state structure obtained from an enzyme catalyzed prenylation reaction and was recently published in the Journal of the American Chemical Society (DOI: 10.1021/ja069119j)

KIE analysis of the prenylation reaction catalyzed by protein-farnesyltransferase. 3D Contour plots of KIEs versus C-O and C-S bond lengths. Left panel: Plot of calculated primary [1-13C] KIE (z-axis, color contours) versus C-O (x-axis) and C-S (y-axis) bond lengths. Right panel: Plot of calculated secondary [1-2H]2 KIE (z-axis, color contours) versus C-O (x-axis) and C-S (y-axis) bond lengths. Center panel: Superposition of calculated contours from Left and Right panels that match the experimentally determined values of the [1-13C] KIE (orange) and [1-2H]2 KIE (green). The region of intersection gives the C-O and C-S bond lengths in the TS.

Transition state structures for nonenzymatic reaction between GPP and ethane thiolate and the corresponding enzyme catalyzed reaction. TS structures were modeled in the gas phase at the m PW1N/6-31G* level of theory. Electrostatic potentials: Left panel, nonenzymatic reaction; Right panel, enzymatic process. Red represents more negative potential (-0.15 au), blue represents less negative potential (-0.05 au) and green is intermediate (-0.10 au). The electrostatic potentials are presented mapped on the 0.005 au isodensity surface.

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