Two tryptophans at the active site of UDP-glucose 4-epimerase from Kluyveromyces fragilis.
Efficient fluorescence energy transfer from aromatic residues to the pyridine moiety of the bound coenzyme (NAD) of UDP-glucose 4-epimerase from Kluyveromyces fragilis had been reported earlier (Mukherji, S., and Bhaduri, A. (1992) J. Biol. Chem. 267, 11709-11713). We have employed N-bromosuccinimide (NBS) to identify tryptophan as the exclusive aromatic donor in the energy transfer. The characteristic UV absorption spectrum associated with Trp oxidation is observed during NBS modification of two of the four Trp residues of native epimerase along with concomitant inactivation of the enzyme. Excellent correlation between the observed inactivation and abolition of fluorescence energy transfer to coenzyme from Trp in epimerase upon treatment with NBS implicates the involvement of the same two tryptophans in both catalytic activity and fluorescence energy transfer. SDS-polyacrylamide gel electrophoresis and fluorescence data preclude gross structural/conformational changes in epimerase due to NBS oxidation. The susceptible tryptophans do not reside at the substrate binding site as substrates and UMP fail to protect against NBS modification. However, failure of sodium borohydride to reduce the bound NAD in the NBS-inactivated epimerase suggests that the reactive tryptophans are close to the coenzyme. Tryptophan fluorescence lifetime values of 1.9 and 3.9 ns for the native and 3.5 ns for the NBS-modified epimerase, complemented by a linear Stern-Volmer plot (effective Stern-Volmer constant = 2.85 M-1) of acrylamide quenching, suggest that the two key tryptophans are buried close to an intrinsic quencher, presumably NAD.
Ray
S
S
Indian Institute of Chemical Biology, Calcutta.
Mukherji
S
S
Bhaduri
A
A
D001665:Binding Sites; D001974:Bromosuccinimide / Q000378:metabolism / Q000494:pharmacology; D004735:Energy Transfer; D007700:Kinetics; D007716:Kluyveromyces / Q000201:enzymology*; D009243:NAD / Q000032:analysis; D013050:Spectrometry, Fluorescence; D014364:Tryptophan; D014534:UDPglucose 4-Epimerase / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism*
D009243:NAD; D014364:Tryptophan; D014534:UDPglucose 4-Epimerase; D001974:Bromosuccinimide