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J. Biol. Chem., Vol. 279, Issue 31, 32796-32803, July 30, 2004

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Determinants of Function and Substrate Specificity in Human UDP-galactose 4'-Epimerase^*

Jenny M. Schulz, Alice L. Watson¶, Rebecca Sanders||, Kerry L. Ross¶, James B. Thoden**, Hazel M. Holden**, and Judith L. Fridovich-Keil¶

From the Graduate Program in Nutrition and Health Sciences, Emory University, ^¶Department of Human Genetics, Emory University School of Medicine, ^||Graduate Program in Genetics and Molecular Biology, Emory University, Atlanta, Georgia 30322 and the ^**Department of Biochemistry, University of Wisconsin, Madison, Wisconsin 53706-1544

UDP-galactose 4'-epimerase (GALE) interconverts UDP-galactose and UDP-glucose in the final step of the Leloir pathway. Unlike the Escherichia coli enzyme, human GALE (hGALE) also efficiently interconverts a larger pair of substrates: UDP-N-acetylgalactosamine and UDP-N-acetylglucosamine. The basis of this differential substrate specificity has remained obscure. Recently, however, x-ray crystallographic data have both predicted essential active site residues and suggested that differential active site cleft volume may be a key factor in determining GALE substrate selectivity. We report here a direct test of this hypothesis. In brief, we have created four substituted alleles: S132A, Y157F, S132A/Y157F, and C307Y-hGALE. While the first three substitutions were predicted to disrupt catalytic activity, the fourth was predicted to reduce active site cleft volume, thereby limiting entry or rotation of the larger but not the smaller substrate. All four alleles were expressed in a null-background strain of Saccharomyces cerevisiae and characterized in terms of activity with regard to both UDP-galactose and UDP-N-acetylgalactosamine. The S132A/Y157F and C307Y-hGALE proteins were also overexpressed in Pichia pastoris and purified for analysis. In all forms tested, the Y157F, S132A, and Y157F/S132A-hGALE proteins each demonstrated a complete loss of activity with respect to both substrates. In contrast, the C307Y-hGALE demonstrated normal activity with respect to UDP-galactose but complete loss of activity with respect to UDP-N-acetylgalactosamine. Together, these results serve to validate the wild-type hGALE crystal structure and fully support the hypothesis that residue 307 acts as a gatekeeper mediating substrate access to the hGALE active site.

Received for publication, May 5, 2004 , and in revised form, June 1, 2004.

^* This work was supported by National Institutes of Health Grant NIDDK R0146403 (to J. L. F.-K.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Supported in part by funds from the Achievement Rewards for College Scientists Foundation, Inc.

To whom correspondence should be addressed: Dept. of Human Genetics, Emory University School of Medicine, Rm. 325.2 Whitehead Bldg., 615 Michael St., Atlanta, GA 30322. Tel.: 404-727-3924; Fax: 404-727-3949; E-mail: jfridov{at}emory.edu.

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