Mutacion Del Q188R

THE JOURNAL OF BIOLOGICAL CHEMISTRY
© 1996 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 271, No. 50, Issue of December 13, pp. 32002–32007, 1996
Printed in U.S.A.

The Q188R Mutation in Human Galactose-1-phosphate
Uridylyltransferase Acts as a Partial Dominant Negative*
(Received for publication, July 11, 1996, and in revised form, September 25, 1996)

J.Patrick Elsevier‡ and Judith L. Fridovich-Keil§¶
From the ‡Graduate Program in Biochemistry and Molecular Biology and the §Department of Genetics and Molecular
Medicine, Emory University, Atlanta, Georgia 30322

Impairment of the human enzyme galactose-1-phosphate
uridylyltransferase (GALT)1 results in the potentially lethal
inborn error of metabolism, galactosemia (2). Normal GALT
catalyzesthe second step of the Leloir pathway of galactose
metabolism, as indicated: UDP-glucose
galactose-1-phosphate 3 UDP-galactose
glucose-1-phosphate (2). GALT enzymes from a variety of sources, including bacteria, yeast, and
human tissues have been isolated and studied in great detail
(2), demonstrating that this enzyme functions with ping-pong
kinetics and a double displacement mechanism ofcatalysis
proceeding through a uridyl-enzyme intermediate (3– 6). The
active site nucleophile for the bacterial enzyme has been identified as histidine 166 (7), which corresponds by homology to
histidine 186 in the human sequence.
Purified GALT enzymes from bacteria (8), yeast (9), and
humans (10) have been shown by a variety of methods to exist

* This work was supported by Grant DK46403from the National
Institutes of Health and a grant from the Emory University Research
Committee (both 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.
¶ To whom correspondence should be addressed:Dept. of Genetics
and Molecular Medicine, Emory University, 1462 Clifton Rd., NE, Atlanta, GA 30322. Tel: 404-727-3924; Fax: 404-727-3949.
1
The abbreviations used are: GALT, galactose-1-phosphate uridylyltransferase; WT, wild type; UDPG, UDP-glucose.

as dimers composed of identical subunits. Furthermore, as
demonstrated by Frey and colleagues from their work with the
Escherichia coli enzyme(11), each GALT subunit contains its
own active site. Recently Wedekind and colleagues (12) confirmed this point when they reported the three-dimensional
structure of the E. coli enzyme refined to 1.8 Å resolution.
That GALT functions as a dimer raises questions of both
fundamental and clinical significance regarding the relationship between dimerization and activity and the impact ofnaturally occurring mutations on both. Indeed, the allelic heterogeneity observed in patient samples (13) demonstrates that
many if not most patients with classic galactosemia are not
true molecular homozygotes but rather compound heterozygotes. This observation raises the possibility that allelic combination and not just allelic constitution may play some role in
determining GALT holoenzyme functionand thereby patient
outcome.
Previously, we have reported the development of a yeast
expression system for human GALT and applied this system to
the study of a handful of patient mutations in the homozygous,
heterozygous, and compound heterozygous states (14 –16). Recently, we have extended this system to include the coexpression of epitope-tagged alleles of GALT, thereby enabling bothstructural and functional studies of specific subunits in the
context of their various dimer states (1). In particular, we have
coexpressed wild-type human GALT with each of two naturally
occurring mutant alleles, Q188R and R333W, and asked the
questions 1) do heterodimers form? and 2) are these heterodimers active? Q188R, which accounts for 60 –70% of the
mutant GALT alleles identified in...