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Deuterated D7-cholesterol analogues as mechanistic probes for wild-type and mutated D7-sterol-C5(6)-desaturase


, : Deuterated D7-cholesterol analogues as mechanistic probes for wild-type and mutated D7-sterol-C5(6)-desaturase. Biochemistry (American Chemical Society) 40(1): 6-67

Deuterium-labeled 5a-cholest-7-en-3b-ol (1) bearing one or two deuteriums at the C-5a and (or) C-6a positions was synthesized in high isotopic and chiral purity. These compounds were used as substrates with the microsomal wild-type Zea mays and recombinant Arabidopsis thaliana D7-sterol-C5-(6)-desaturases (5-DES) to probe directly the stereochemistry and the mechanism of the enzymatic reaction. Clearly, in the conversion of 1 by both 5-DESs, the 6a-hydrogen is removed. [6a-2H]-5a-Cholest-7-en-3b-ol shows an intermolecular deuterium kinetic isotope effect (DKIE) on V and V/K, D6V = 2.6 [plus or minus] 0.3, D6V/K = 2.4 [plus or minus] 0.1; and D6V = 2.3 [plus or minus] 0.3, D6V/K = 2.3 [plus or minus] 0.2 for the Zea mays and A. thaliana wild-type 5-DES, respectively. In contrast, negligible or minor isotope effects, D5V = 0.99 [plus or minus] 0.04, D5V/K = 0.91 [plus or minus] 0.08; and D5V = 0.93 [plus or minus] 0.06, D5V/K = 0.96 [plus or minus] 0.04, respectively, were observed with [5a-2H]-cholest-7-en-3b-ol. The observed pattern of isotope effects strongly suggests that the plant 5-DES initiates oxidation by cleavage of the chemically activated C6a-H bond, a step which appears to be partially rate-limiting in the desaturation process. Cleavage of the C5--H bond has a negligible isotope effect, indicating that the desaturation involves asynchronous 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 essential to maintaining desaturase activity, although V/K values for mutant T114I and T114S were respectively 10-fold lower and 4-fold higher than that of the native 5-DES. In this study, we combined variation in enzyme structure and DKIE studies and showed that D6V and D6V/K increased respectively to 3.8 [plus or minus] 0.3 and 3.8 [plus or minus] 0.4 in mutant T114I and decreased respectively to 1.6 [plus or minus] 0.4 and 1.7 [plus or minus] 0.1 in mutant T114S. The data suggest that the conserved hydroxyl function at position 114 in the ERG3 family makes the abstraction of the 6a-hydrogen atom substantially less rate-limiting during the 5-DES reaction. Based on the data, a tentative mechanism for the desaturation of cholest-7-en-3b-ol is proposed. Reprinted by permission of the publisher.

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