Jb. Thoden et al., Crystallographic evidence for Tyr 157 functioning as the active site base in human UDP-galactose 4-epimerase, BIOCHEM, 39(19), 2000, pp. 5691-5701
UDP-galactose 4-epimerase catalyzes the interconversion of UDP-glucose and
UDP-galactose during normal galactose metabolism. In humans, deficiencies i
n this enzyme lead to the complex disorder referred to as epimerase-deficie
ncy galactosemia. Here, we describe the high-resolution X-ray crystallograp
hic structures of human epimerase in the resting state (i.e., with bound NA
D(+)) and in a ternary complex with bound NADH and UDP-glucose. Those amino
acid side chains responsible for anchoring the NAD(+) to the protein inclu
de Asp 33, Asn 37, Asp 66, Tyr 157, and Lys 161. The glucosyl group of the
substrate is bound to the protein via the side-chain carboxamide groups of
Asn 187 and Asn 207. Additionally, O-gamma of Ser 132 and O-eta of Tyr 157
lie within 2.4 and 3.1 Angstrom, respectively, of the 4'-hydroxyl group of
the sugar. Comparison of the polypeptide chains for the resting enzyme and
for the protein with bound NADH and UDP-glucose demonstrates that the major
conformational changes which occur upon substrate binding are limited prim
arily to the regions defined by Glu 199 to Asp 240 and Gly 274 to Tyr 308,
Additionally, this investigation reveals for the first time that a conserve
d tyrosine, namely Tyr 157, is in the proper position to interact directly
with the 4'-hydroxyl group of the sugar substrate and to thus serve as the
active-site base. A low barrier hydrogen bond between the 4'-hydroxyl group
of the sugar and Or of Ser 132 facilitates proton transfer from the sugar
4'-hydroxyl group to O-eta of Tyr 157.