Distinct functional roles of two active site thiols in UDPglucose 4-epimerase from Kluyveromyces fragilis.
UDPglucose 4-epimerase from Kluyveromyces fragilis was earlier shown to have two conformationally vicinal thiols at the active site. Upon treatment with diamide, these thiols form a disulfide linkage across the subunits that results in coordinated loss of catalytic activity and coenzyme fluorescence (Ray, M., and Bhaduri, A. (1980) J. Biol. Chem. 255, 10777-10786). Employing a number of thiol-specific reagents, we now suggest discriminatory and nonidentical roles for these two thiols. Kinetic and statistical analysis of 5,5'-dithiobis-(2-nitrobenzoic acid) and N-ethylmaleimide modification reaction of epimerase show that only one thiol is essential for activity. Consecutive modification experiments clearly show that the same active thiol is modified in both cases. However, significant differences are observed when the reactivity of these reagents is monitored in terms of coenzyme fluorescence. Treatment with N-ethylmaleimide leads to a form of inactive enzyme that fully retains its fluorescent properties whereas modification with 5,5'-dithiobis-(2-nitrobenzoic acid), on the other hand, results in the loss of both activity and fluorescence. The closely spaced nonessential second thiol, which is not modified by N-ethylmaleimide is therefore involved in generating and maintaining the coenzyme fluorescence. Modification studies with a series of spin-labeled maleimide shows that only 3-(maleimidomethyl)proxyl causes partial quenching of coenzyme fluorescence. This suggests that the active thiol is situated at a distance of 4.5 A approximately from the coenzyme fluorophore.
Bhattacharjee
H
H
Indian Institute of Chemical Biology, Calcutta.
Bhaduri
A
A
D001665:Binding Sites; D002384:Catalysis; D003067:Coenzymes / Q000378:metabolism; D004228:Dithionitrobenzoic Acid / Q000494:pharmacology; D005033:Ethylmaleimide / Q000494:pharmacology; D007716:Kluyveromyces / Q000201:enzymology*; D013050:Spectrometry, Fluorescence; D013113:Spin Labels; D013438:Sulfhydryl Compounds / Q000378:metabolism*; D014534:UDPglucose 4-Epimerase / Q000037:antagonists & inhibitors / Q000378:metabolism*
D003067:Coenzymes; D013113:Spin Labels; D013438:Sulfhydryl Compounds; D004228:Dithionitrobenzoic Acid; D014534:UDPglucose 4-Epimerase; D005033:Ethylmaleimide