A 17-amino acid insert changes UDP-N-acetylhexosamine pyrophosphorylase specificity from UDP-GalNAc to UDP-GlcNAc.
We previously reported the purification of a UDP-N-acetylhexosamine (UDP-HexNAc) pyrophosphorylase from pig liver that catalyzed the synthesis of both UDP-GlcNAc and UDP-GalNAc from UTP and the appropriate HexNAc-1-P (Szumilo, T., Zeng, Y., Pastuszak, I., Drake, R., Szumilo, H., and Elbein, A. D. (1996) J. Biol. Chem. 271, 13147-13154). Both sugar nucleotides were synthesized at nearly the same rate, although the Km for GalNAc-1-P was about 3 times higher than for GlcNAc-1-P. Based on native gels and SDS-polyacrylamide gel electrophoresis, the enzyme appeared to be a dimer of 120 kDa composed of two subunits of about 57 and 64 kDa. Three peptides sequenced from the 64-kDa protein and two from the 57-kDa protein showed 100% identity to AGX1, a 57-kDa protein of unknown function from human sperm. An isoform called AGX2 is identical in sequence to AGX1 except that it has a 17-amino acid insert near the carboxyl terminus. We expressed the AGX1 and AGX2 genes in Escherichia coli. The protein isolated from the AGX1 clone comigrated on SDS gels with the liver 57-kDa pyrophosphorylase subunit and was 2-3 times more active with GalNAc-1-P than with GlcNAc-1-P. On the other hand, the protein from the AGX2 clone migrated with the liver 64-kDa pyrophosphorylase subunit and had 8-fold better activity with GlcNAc-1-P than with GalNAc-1-P. These results indicate that insertion of the 17-amino acid peptide modifies the specificity of the pyrophosphorylase from synthesis of UDP-GalNAc to synthesis of UDP-GlcNAc.
Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, Little Rock, Arkansas 72205, USA.
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Wang-Gillam
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Pastuszak
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Elbein
D000116:Acetylgalactosamine / Q000031:analogs & derivatives / Q000378:metabolism; D000117:Acetylglucosamine / Q000031:analogs & derivatives / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D019281:Dimerization; D006801:Humans; D008099:Liver / Q000201:enzymology; D008969:Molecular Sequence Data; D009713:Nucleotidyltransferases / Q000378:metabolism*; D013379:Substrate Specificity; D013552:Swine; D014536:Uridine Diphosphate N-Acetylgalactosamine / Q000378:metabolism*; D014537:Uridine Diphosphate N-Acetylglucosamine / Q000378:metabolism*
C115144:N-acetylgalactosamine-1-phosphate; D014537:Uridine Diphosphate N-Acetylglucosamine; C026146:N-acetylglucosamine-1-phosphate; D014536:Uridine Diphosphate N-Acetylgalactosamine; D009713:Nucleotidyltransferases; C023226:UDPacetylglucosamine pyrophosphorylase; D000116:Acetylgalactosamine; D000117:Acetylglucosamine