Deuterated Delta(7)-cholestenol analogues as mechanistic probes for wild-type and mutated Delta(7)-sterol-C5(6)-desaturase

Deuterium-labeled 5 alpha -cholest-7-en-3 beta -ol (1) bearing one or two d euteriums at the C-5 alpha and (or) C-6 alpha positions was synthesized in high isotopic and chiral purity. These compounds were used as substrates wi th the microsomal wild-type Zea mays and recombinant Arabidopsis thaliana D elta (7)-sterol-C5-(6)-desaturases (5-DES) to probe directly the stereochem istry and the mechanism of the enzymatic reaction. Clearly, in the conversi on of I by both 5-DESs, the 6 alpha -hydrogen is removed. [6 alpha-H-2]-5 a lpha -Cholest-7-en-3 beta -ol shows an intermolecular deuterium kinetic iso tope effect (DKIE) on V and V/K, V-D6 = 2.6 +/- 0.3, V-D6/K = 2.4 +/- 0.1; and V-D6 = 2.3 +/- 0.3, V-D6/K = 2.3 +/- 0.2 for the Zea mays and A. thalia na wild-type 5-DES, respectively. In contrast, negligible or minor isotope effects, V-D5 = 0.99 +/- 0.04, V-D5/K = 0.91 +/- 0.08; and V-D5 = 0.93 +/- 0.06, V-D5/K = 0.96 +/- 0.04, respectively, were observed with [5 alpha-H-2 ]-cholest-7-en-3 beta -ol. The observed pattern of isotope effects strongly suggests that the plant 5-DES initiates oxidation by cleavage of the chemi cally activated C6 alpha -H bond, a step which appears to be partially rate -limiting in the desaturation process. Cleavage of the C5-H bond has a negl igible isotope effect, indicating that the desaturation involves asynchrono us scission of the two C-H bonds at C5 and C6. We showed previously [Taton, M., et al. (2000) Biochemistry 39, 701] that threonine 114 was not essenti al to maintaining desaturase activity, although V/K values for mutant T114I and T114S were respectively 10-fold lower and 3-fold higher than that of t he native 5-DES. In this study, we combined variation in enzyme structure a nd DKIE studies and showed that V-D6 and V-D6/K increased respectively to 3 .8 +/- 0.3 and 3.8 +/- 0.4 in mutant T114I and decreased respectively to 1. 6 +/- 0.3 and 1.7 +/- 0.1 ill mutant T114S. The data suggest that the conse rved hydroxyl function at position 114 in the ERG3 family makes the abstrac tion of the 6 alpha -hydrogen atom substantially less rate-limiting during the 5-DES reaction. Based on the data, a tentative mechanism for the desatu ration of cholest-7-en-3 beta -ol is proposed.