Identification and Modification of the Uridine-binding Site of the UDP-GalNAc (GlcNAc) Pyrophosphorylase

Abstract

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. 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. UDP-GalNAc pyrophosphorylase N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine polyacrylamide gel electrophoresis human sarcoma virus type 1 thymidine kinase 3′,5′-diazido-2′,3′-dideoxyuridine [32P]5′-monophosphate UDP-GlcNAc pyrophosphorylase (UDP-GlcNAcPP)1 is a key enzyme in the biosynthesis of complex carbohydrates, since it is the enzyme that produces the activated form of GlcNAc, i.e.UDP-GlcNAc, for polymerization. This enzyme catalyzes the reversible reaction: UTP + GlcNAc-1-P ⇌ UDP-GlcNAc + PPi. The enzyme was initially purified about 100-fold from Staphylococcus aureus, and that enzyme preparation utilized UDP-GalNAc (in the reverse direction) at about 2.8% the rate of UDP-GlcNAc (1.Strominger J.L. Smith M.S. J. Biol. Chem. 1959; 234: 1822-1827Abstract Full Text PDF PubMed Google Scholar). The enzyme was also partially purified from calf brain, and in that case, the enzyme also utilized UDP-glucose at about 36% the rate of UDP-GlcNAc (2.Pattabiraman T.N. Bachhawat B.K. Biochim. Biophys. Acta. 1961; 50: 129-134Crossref PubMed Scopus (17) Google Scholar). However, it was not clear from that report whether the calf brain enzyme had any activity with UDP-GalNAc, or whether the activity with UDP-glucose was due to the same enzyme, or to a contaminating pyrophosphorylase.We purified the UDP-GlcNAcPP from pig liver cytosol to apparent homogeneity, and found that the purified enzyme contained two proteins with molecular masses of about 64 and 57 kDa. Interestingly enough, this purified enzyme also catalyzed the synthesis of UDP-GalNAc from UTP and GalNAc-1-P, and at saturating concentrations of GalNAc-1-P, the rate of synthesis of UDP-GalNAc was equal to that of UDP-GlcNAc (3.Szumilo T. Zeng Y. Pastuszak I. Drake R.R. Szumilo H. Elbein A.D. J. Biol. Chem. 1996; 271: 13147-13154Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar). We sequenced three peptides from the 64-kDa protein and two from the 57-kDa protein and found that all of these sequences showed 100% identity to AGX1, a protein of unknown function from the sperm of infertile males (4.Diekman A.B. Goldberg E. Biol. Reprod. 1994; 50: 1087-1097Crossref PubMed Scopus (39) Google Scholar). A second human protein of wider tissue distribution, but also of unknown function and called AGX2, has the identical sequence to AGX1 except that AGX2 has an additional 17-amino acid insert near the carboxyl terminus (5.Wang-Gillam A. Pastuszak I. Elbein A.D. J. Biol. Chem. 1998; 273: 27055-27057Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar).We cloned the cDNAs for AGX1 and AGX2 and expressed the active proteins in Escherichia coli. AGX1 migrated with the 57-kDa pig liver enzyme on SDS gels, and had about three times as much UDP-GalNAc synthetic activity as UDP-GlcNAc activity. On the other hand, AGX2 migrated with the 64-kDa pig liver protein and also had both UDP-GalNAc and UDP-GlcNAc activity, but at a ratio of about 1:8 (5.Wang-Gillam A. Pastuszak I. Elbein A.D. J. Biol. Chem. 1998; 273: 27055-27057Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). Thus, addition of the 17-amino acid insert changes the activity of the enzyme from synthesizing UDP-GalNAc to synthesizing UDP-GlcNAc (5.Wang-Gillam A. Pastuszak I. Elbein A.D. J. Biol. Chem. 1998; 273: 27055-27057Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar).In the current study, we have identified a 24-amino acid peptide involved in binding the uridine portion of the substrate,i.e. either UDP-GalNAc or UDP-GlcNAc. To do this, we synthesized N3-UDP-[32P]GlcNAc and N3-UDP-[32P]GalNAc (6.Drake R.R. Elbein A.D. Glycobiology. 1992; 2: 279-284Crossref PubMed Scopus (27) Google Scholar, 7.Sunthankar P. Pastuszak I. Rooke A. Elbein A.D. Rijn I. Canfield W. Drake R.R. Anal. Biochem. 1998; 258: 195-201Crossref PubMed Scopus (13) Google Scholar), and incubated both of these photoprobes with the recombinant AGX1 (i.e.UDPGalNAcPP). Both photoprobes cross-linked specifically to amino acids in the active site, and the resulting radiolabeled peptide was identified and located in the protein sequence of AGX1. We then used this information to do site-directed mutagenesis of four highly conserved amino acids, and determined the effect of amino acid changes on enzymatic activity and substrate binding. These studies indicated that two glycines in that peptide (at positions 222 and 224) are important in cross-linking the uridine portion of the substrate, as well as in enzyme catalysis. These glycines may play a direct role in binding and catalysis, or they may be important in protein conformation.Another study had previously reported the identity between AGX1 and the UDP-GlcNAc pyrophosphorylase (8.Mio T. Yabe T. Arisawa M. Yamada-Okabe H. J. Biol. Chem. 1998; 273: 14392-14397Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar). That study indicated that Gly112, Arg116, and Lys123 were important for catalytic activity of this enzyme. We also confirmed that changing Gly111 (position 111 in our sequence) to Ala resulted in loss of catalytic activity, but importantly, this modification did not eliminate the ability of the mutant protein to cross-link the photoprobes. These studies begin to identify important amino acid residues and peptide sequences involved in substrate binding and catalysis. UDP-GlcNAc pyrophosphorylase (UDP-GlcNAcPP)1 is a key enzyme in the biosynthesis of complex carbohydrates, since it is the enzyme that produces the activated form of GlcNAc, i.e.UDP-GlcNAc, for polymerization. This enzyme catalyzes the reversible reaction: UTP + GlcNAc-1-P ⇌ UDP-GlcNAc + PPi. The enzyme was initially purified about 100-fold from Staphylococcus aureus, and that enzyme preparation utilized UDP-GalNAc (in the reverse direction) at about 2.8% the rate of UDP-GlcNAc (1.Strominger J.L. Smith M.S. J. Biol. Chem. 1959; 234: 1822-1827Abstract Full Text PDF PubMed Google Scholar). The enzyme was also partially purified from calf brain, and in that case, the enzyme also utilized UDP-glucose at about 36% the rate of UDP-GlcNAc (2.Pattabiraman T.N. Bachhawat B.K. Biochim. Biophys. Acta. 1961; 50: 129-134Crossref PubMed Scopus (17) Google Scholar). However, it was not clear from that report whether the calf brain enzyme had any activity with UDP-GalNAc, or whether the activity with UDP-glucose was due to the same enzyme, or to a contaminating pyrophosphorylase. We purified the UDP-GlcNAcPP from pig liver cytosol to apparent homogeneity, and found that the purified enzyme contained two proteins with molecular masses of about 64 and 57 kDa. Interestingly enough, this purified enzyme also catalyzed the synthesis of UDP-GalNAc from UTP and GalNAc-1-P, and at saturating concentrations of GalNAc-1-P, the rate of synthesis of UDP-GalNAc was equal to that of UDP-GlcNAc (3.Szumilo T. Zeng Y. Pastuszak I. Drake R.R. Szumilo H. Elbein A.D. J. Biol. Chem. 1996; 271: 13147-13154Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar). We sequenced three peptides from the 64-kDa protein and two from the 57-kDa protein and found that all of these sequences showed 100% identity to AGX1, a protein of unknown function from the sperm of infertile males (4.Diekman A.B. Goldberg E. Biol. Reprod. 1994; 50: 1087-1097Crossref PubMed Scopus (39) Google Scholar). A second human protein of wider tissue distribution, but also of unknown function and called AGX2, has the identical sequence to AGX1 except that AGX2 has an additional 17-amino acid insert near the carboxyl terminus (5.Wang-Gillam A. Pastuszak I. Elbein A.D. J. Biol. Chem. 1998; 273: 27055-27057Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). We cloned the cDNAs for AGX1 and AGX2 and expressed the active proteins in Escherichia coli. AGX1 migrated with the 57-kDa pig liver enzyme on SDS gels, and had about three times as much UDP-GalNAc synthetic activity as UDP-GlcNAc activity. On the other hand, AGX2 migrated with the 64-kDa pig liver protein and also had both UDP-GalNAc and UDP-GlcNAc activity, but at a ratio of about 1:8 (5.Wang-Gillam A. Pastuszak I. Elbein A.D. J. Biol. Chem. 1998; 273: 27055-27057Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). Thus, addition of the 17-amino acid insert changes the activity of the enzyme from synthesizing UDP-GalNAc to synthesizing UDP-GlcNAc (5.Wang-Gillam A. Pastuszak I. Elbein A.D. J. Biol. Chem. 1998; 273: 27055-27057Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). In the current study, we have identified a 24-amino acid peptide involved in binding the uridine portion of the substrate,i.e. either UDP-GalNAc or UDP-GlcNAc. To do this, we synthesized N3-UDP-[32P]GlcNAc and N3-UDP-[32P]GalNAc (6.Drake R.R. Elbein A.D. Glycobiology. 1992; 2: 279-284Crossref PubMed Scopus (27) Google Scholar, 7.Sunthankar P. Pastuszak I. Rooke A. Elbein A.D. Rijn I. Canfield W. Drake R.R. Anal. Biochem. 1998; 258: 195-201Crossref PubMed Scopus (13) Google Scholar), and incubated both of these photoprobes with the recombinant AGX1 (i.e.UDPGalNAcPP). Both photoprobes cross-linked specifically to amino acids in the active site, and the resulting radiolabeled peptide was identified and located in the protein sequence of AGX1. We then used this information to do site-directed mutagenesis of four highly conserved amino acids, and determined the effect of amino acid changes on enzymatic activity and substrate binding. These studies indicated that two glycines in that peptide (at positions 222 and 224) are important in cross-linking the uridine portion of the substrate, as well as in enzyme catalysis. These glycines may play a direct role in binding and catalysis, or they may be important in protein conformation. Another study had previously reported the identity between AGX1 and the UDP-GlcNAc pyrophosphorylase (8.Mio T. Yabe T. Arisawa M. Yamada-Okabe H. J. Biol. Chem. 1998; 273: 14392-14397Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar). That study indicated that Gly112, Arg116, and Lys123 were important for catalytic activity of this enzyme. We also confirmed that changing Gly111 (position 111 in our sequence) to Ala resulted in loss of catalytic activity, but importantly, this modification did not eliminate the ability of the mutant protein to cross-link the photoprobes. These studies begin to identify important amino acid residues and peptide sequences involved in substrate binding and catalysis.