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J Biol Chem, Vol. 275, Issue 2, 1433-1438, January 14, 2000
From the Department of Biochemistry and Molecular Biology,
University of Arkansas for Medical Sciences,
Little Rock, Arkansas 72205
UDP-GalNAc pyrophosphorylase (UDP-GalNAcPP; AGX1)
catalyzes the synthesis of UDP-GalNAc from UTP and GalNAc-1-P. The
475-amino acid protein (57 kDa protein) also synthesizes UDP-GlcNAc at
about 25% the rate of UDP-GalNAc. The cDNA for this enzyme, termed
AGX1, was cloned in Escherichia coli, and expressed as an
active enzyme that cross-reacted with antiserum against the original
pig liver UDP-HexNAcPP. In the present study, we incubated recombinant
AGX1 with N3-UDP-[32P]GlcNAc and
N3-UDP-[32P]GalNAc probes to label the
nucleotide-binding site. Proteolytic digestions of the labeled enzyme
and analysis of the resulting peptides indicated that both photoprobes
cross-linked to one 24-amino acid peptide located between residues
Val216 and Glu240. Four amino acids in this
peptide were found to be highly conserved among closely related
enzymes, and each of these was individually modified to alanine.
Mutation of Gly222 to Ala in the peptide almost completely
eliminated UDP-GlcNAc and UDP-GalNAc synthesis, while mutation of
Gly224 to Ala, almost completely eliminated UDP-GalNAc
synthesis, but UDP-GlcNAc was only diminished by 50%. Both of these
mutations also resulted in almost complete loss of the ability of the
mutated proteins to cross-link
N3-UDP-[32P]GlcNAc or
N3-UDP-[32P]GalNAc. On the other hand,
mutations of either Pro220 or Tyr227 to Ala did
not greatly affect enzymatic activity, although there was some
reduction in the ability of these proteins to cross-link the
photoaffinity probes. We also mutated Gly111 to Ala since
this amino acid was reported to be necessary for catalysis (Mio, T.,
Yabe, T., Arisawa, M., and Yamada-Okabe, H. (1998) J. Biol.
Chem. 273, 14392-14397). The Gly111 to Ala mutant
lost all enzymatic activity, but interestingly enough, this mutant
protein still cross-linked the radioactive N3-UDP-GlcNAc
although not nearly as well as the wild type. On the other hand,
mutation of Arg115 to Ala had no affect on enzymatic
activity although it also reduced the amount of cross-linking of
N3-UDP-[32P]GlcNAc. These studies help to
define essential amino acids at or near the nucleotide-binding site and
the catalytic site, as well as peptides involved in binding and catalysis.
Identification and Modification of the Uridine-binding Site of
the UDP-GalNAc (GlcNAc) Pyrophosphorylase*
*
This work was supported by National Institutes of Health
Grant HL-17783 (to A. D. E.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The 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 Biochemistry
and Molecular Biology, University of Arkansas for Medical Sciences,
Slot 516, 4301 West Markham, Little Rock, AR 72205-7199. Tel.:
501-686-5196; Fax: 501-686-8169; E-mail:
elbeinaland@exchange.uams.edu.
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