Kinetics of the Reductive Half-Reaction of the Iron-Sulfur Flavoenzyme CDP-6-deoxy-L-THREO-D-glycero-4-hexulose-3-dehydrase Reductase
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- 作者：George T. Gassner ; David A. Johnson ; Hung-wen Liu ; and David P. Ballou
- 刊名：Biochemistry
- 出版年：1996
- 出版时间：June 18, 1996
- 年：1996
- 卷：35
- 期：24
- 页码：7752 - 7761
- 全文大小：531K
- 年卷期：v.35,no.24(June 18, 1996)
- ISSN：1520-4995
文摘
The conversion ofCDP-4-keto-6-deoxy-D-glucose toCDP-4-keto-3,6-dideoxy-D-glucose is akey step in biosynthesis of ascarylose, the terminal dideoxyhexose ofthe O-antigen tetrasaccharide of thelipopolysaccharide from Yersinia pseudotuberculosis V.This transformation is catalyzed by twoenzymes:CDP-6-deoxy-L-threo-D-glycero-4-hexulose-3-dehydrase(E1), which contains a pyridoxamineand a [2Fe-2S] center, and an NADH-dependentCDP-6-deoxy-L-threo-D-glycero-4-hexulose-3-dehydrasereductase (E3), which contains both an FAD and a [2Fe-2S]center. E1 reacts to form a Schiff basewithCDP-4-keto-6-deoxy-D-glucose and catalyzes the eliminationof the hydroxyl at position 3 of the glucosemoiety, resulting in the formation of a covalently boundCDP-6-deoxy-
3,4-glucoseen intermediate.E3transfers electrons from NADH to E1, which uses these toreduce the 3,4-glucoseen bond to produceCDP-4-keto-3,6-dideoxy-D-glucose. In this work, wehave investigated the reductive half-reaction ofE3using both single wavelength and diode array stopped flow absorbancespectroscopy. We find that NADHbinds to both oxidized (Kd = 52.5 ± 2 
M)and two-electron-reduced (Kd = 12.1 ± 1M) forms of E3.Hydride transfer from NADH to the FAD moiety occurs at 107.5 ± 3s-1 and exhibits a 10-fold deuteriumisotope effect when (4R)-[2H]NADH issubstituted for NADH. Following the hydride transferreaction,NAD+ is released at 42.5 ± 1 s-1 andelectron transfer from the reduced FAD to the [2Fe-2S]centeroccurs rapidly. The extent of the intramolecular electron transferreaction is pH-dependent with a pKaof7.3 ± 0.1, which may represent the ionization state of the N-1position of the FAD hydroquinone of E3.Finally, E3 is converted to the three-electron-reducedstate in a slow disproportionation reaction thatconsumes NADH. The [2Fe-2S] center of E3 wasselectively disassembled by titration with mersalyl togive E3(apoFeS). The properties of this form ofthe enzyme are compared to those of the holoenzyme.Similarities and differences of the reductive half-reactions ofE3 and related iron-sulfur flavoenzymesare discussed.
3,4-glucoseen intermediate.E3transfers electrons from NADH to E1, which uses these toreduce the 3,4-glucoseen bond to produceCDP-4-keto-3,6-dideoxy-D-glucose. In this work, wehave investigated the reductive half-reaction ofE3using both single wavelength and diode array stopped flow absorbancespectroscopy. We find that NADHbinds to both oxidized (Kd = 52.5 ± 2 M)and two-electron-reduced (Kd = 12.1 ± 1M) forms of E3.Hydride transfer from NADH to the FAD moiety occurs at 107.5 ± 3s-1 and exhibits a 10-fold deuteriumisotope effect when (4R)-[2H]NADH issubstituted for NADH. Following the hydride transferreaction,NAD+ is released at 42.5 ± 1 s-1 andelectron transfer from the reduced FAD to the [2Fe-2S]centeroccurs rapidly. The extent of the intramolecular electron transferreaction is pH-dependent with a pKaof7.3 ± 0.1, which may represent the ionization state of the N-1position of the FAD hydroquinone of E3.Finally, E3 is converted to the three-electron-reducedstate in a slow disproportionation reaction thatconsumes NADH. The [2Fe-2S] center of E3 wasselectively disassembled by titration with mersalyl togive E3(apoFeS). The properties of this form ofthe enzyme are compared to those of the holoenzyme.Similarities and differences of the reductive half-reactions ofE3 and related iron-sulfur flavoenzymesare discussed.