title: Kinetic and mechanistic properties of biotin sulfoxide reductase.
abstract: Rhodobacter sphaeroides f. sp. denitrificans biotin sulfoxide reductase catalyzes the reduction of d-biotin d-sulfoxide (BSO) to biotin. Initial rate studies of the homogeneous recombinant enzyme, expressed in Escherichia coli, have demonstrated that the purified protein utilizes NADPH as a facile electron donor in the absence of any additional auxiliary proteins. We have previously shown [Pollock, V. V., and Barber, M. J. (1997) J. Biol. Chem. 272, 3355-3362] that, at pH 8 and in the presence of saturating concentrations of BSO, the enzyme exhibits, a marked preference for NADPH (k(cat,app) = 500 s(-1), K(m,app) = 269 microM, and k(cat,app)/K(m,app) = 1.86 x 10(6) M(-1) s(-1)) compared to NADH (k(cat,app) = 47 s(-1), K(m,app) = 394 microM, and k(cat,app)/K(m,app) = 1.19 x 10(5) M(-1) s(-1)). Production of biotin using NADPH as the electron donor was confirmed by both the disk biological assay and by reversed-phase HPLC analysis of the reaction products. The purified enzyme also utilized ferricyanide as an artificial electron acceptor, which effectively suppressed biotin sulfoxide reduction and biotin formation. Analysis of the enzyme isolated from tungsten-grown cells yielded decreased reduced methyl viologen:BSO reductase, NADPH:BSO reductase, and NADPH:FR activities, confirming that Mo is required for all activities. Kinetic analyses of substrate inhibition profiles revealed that the enzyme followed a Ping Pong Bi-Bi mechanism with both NADPH and BSO exhibiting double competitive substrate inhibition. Replots of the 1/v-axes intercepts of the parallel asymptotes obtained at several low concentrations of fixed substrate yielded a K(m) for BSO of 714 and 65 microM for NADPH. In contrast, utilizing NADH as an electron donor, the replots yielded a K(m) for BSO of 132 microM and 1.25 mM for NADH. Slope replots of data obtained at high concentrations of BSO yielded a K(i) for BSO of 6.10 mM and 900 microM for NADPH. Kinetic isotope studies utilizing stereospecifically deuterated NADPD indicated that BSO reductase uses specifically the 4R-hydrogen of the nicotinamide ring. Cyanide inhibited NADPH:BSO and NADPH:FR activities in a reversible manner while diethylpyrocarbonate treatment resulted in complete irreversible inactivation of the enzyme concomitant with molybdenum cofactor release, indicating that histidine residues are involved in cofactor-binding.
authors.affiliation: Department of Biochemistry and Molecular Biology, College of Medicine University of South Florida, Tampa, Florida 33612, USA.
authors.forename: V V
authors.initials: VV
authors.lastname: Pollock
authors.forename: M J
authors.initials: MJ
authors.lastname: Barber
mesh_terms: D001431:Bacteriological Techniques; D001667:Binding, Competitive; D002851:Chromatography, High Pressure Liquid; D003067:Coenzymes / Q000737:chemistry; D003903:Deuterium / Q000737:chemistry; D004047:Diethyl Pyrocarbonate / Q000737:chemistry; D004789:Enzyme Activation; D006150:Guanine Nucleotides / Q000737:chemistry; D007700:Kinetics; D008667:Metalloproteins / Q000737:chemistry; D000090006:Molybdenum Cofactors; D009249:NADP / Q000737:chemistry; D010088:Oxidoreductases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000302:isolation & purification; D011190:Potassium Cyanide / Q000737:chemistry; D011621:Pteridines / Q000737:chemistry; D011622:Pterins / Q000737:chemistry; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000302:isolation & purification; D012242:Rhodobacter sphaeroides / Q000201:enzymology* / Q000254:growth & development; D013379:Substrate Specificity; D014414:Tungsten / Q000032:analysis
chemical_list: D003067:Coenzymes; D003903:Deuterium; D004047:Diethyl Pyrocarbonate; D006150:Guanine Nucleotides; D008667:Metalloproteins; D000090006:Molybdenum Cofactors; D009249:NADP; D010088:Oxidoreductases; D011190:Potassium Cyanide; D011621:Pteridines; D011622:Pterins; D011994:Recombinant Proteins; D014414:Tungsten; C063761:molybdopterin guanine dinucleotide; C022261:molybdopterin; C019792:biotin sulfoxide reductase