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.