Deuterated delta 7-cholestenol analogues as mechanistic probes for wild-type and mutated delta 7-sterol-C5(6)-desaturase.
Deuterium-labeled 5alpha-cholest-7-en-3beta-ol (1) bearing one or two deuteriums at the C-5alpha and (or) C-6alpha 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 Delta(7)-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 6alpha-hydrogen is removed. [6alpha-(2)H]-5alpha-Cholest-7-en-3beta-ol shows an intermolecular deuterium kinetic isotope effect (DKIE) on V and V/K, (D6)V = 2.6+/-0.3, (D6)V/K = 2.4+/-0.1; and (D6)V = 2.3 +/-0.3, (D6)V/K = 2.3+/-0.2 for the Zea mays and A. thaliana wild-type 5-DES, respectively. In contrast, negligible or minor isotope effects, (D5)V = 0.99+/-0.04, (D5)V/K = 0.91+/-0.08; and (D5)V = 0.93 +/-0.06, (D5)V/K = 0.96+/-0.04, respectively, were observed with [5alpha-(2)H]-cholest-7-en-3beta-ol. The observed pattern of isotope effects strongly suggests that the plant 5-DES initiates oxidation by cleavage of the chemically activated C6alpha-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 (D6)V and (D6)V/K increased respectively to 3.8+/-0.3 and 3.8+/-0.4 in mutant T114I and decreased respectively to 1.6+/-0.4 and 1.7+/- 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 6alpha-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-3beta-ol is proposed.
Rahier
A
A
Département Isoprénoïdes, Institut de Biologie Moléculaire des Plantes, CNRS FRE 2161, 28 rue Goethe, 67083 Strasbourg Cédex, France. enzymo@bota-ulp.u-strasbg.fr
D017360:Arabidopsis / Q000201:enzymology / Q000235:genetics; D002384:Catalysis; D003684:Dehydrocholesterols / Q000138:chemical synthesis / Q000737:chemistry*; D003903:Deuterium / Q000737:chemistry*; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D010088:Oxidoreductases / Q000737:chemistry* / Q000235:genetics*; D044925:Oxidoreductases Acting on CH-CH Group Donors; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D013237:Stereoisomerism; D013379:Substrate Specificity / Q000235:genetics; D003313:Zea mays / Q000201:enzymology
D003684:Dehydrocholesterols; D007527:Isoenzymes; C033144:lathosterol delta-5-dehydrogenase; D003903:Deuterium; C016705:7-dehydrocholesterol; D010088:Oxidoreductases; D044925:Oxidoreductases Acting on CH-CH Group Donors