The role of histidine residues in the HXGH site of CTP:phosphocholine cytidylyltransferase in CTP binding and catalysis.
The HXGH motif of CTP:phosphocholine cytidylyltransferase (CCT) is a unifying feature of the cytidylyltransferase family which has been proposed to function in binding of CTP and catalysis [Veitch, D. P. & Cornell, R. B. (1996) Biochemistry 35, 10743-10750]. Substitution of serine for Gly91 in the HXGH motif of CCT implicates this motif in CTP-binding [Park, Y. S., Gee, P., Sanker, S., Schuster, E. J., Zuiderweg, E. R. & Kent, C. (1997) J. Biol. Chem. 272, 15161]. The model for CTP binding involves hydrogen bond contacts between the histidine imidazole and the CTP phosphate oxygens. We have mutated His89 and His92 to Gly or Ala, which eliminate potential hydrogen bonds, and to Asn or Gln, which conserve these interactions. Mutation to Gly or Ala at both positions, and the H89Q mutation resulted in inactive enzymes. The Vmax of [N89]CT was 100-fold lower than that of wild-type CCT, but CTP binding was not perturbed, suggesting an involvement of His89 in transition-state stabilization. The H92N mutation reduced Vmax and increased the Kms for both substrates fivefold. The H92Q mutation had little effect on substrate binding or Vmax. These data suggest that the Gln92 NH2, and not the Asn NH2, is able to substitute for the histidine NH, and implicates the tau nitrogen of His92 in forming contacts with CTP. This work strengthens the hypothesis that the HXGH motif is involved in the binding of CTP and transition-state stabilization.
Veitch
D P
DP
Institute of Molecular Biology and Biochemistry and Department of Chemistry, Simon Fraser University, Burnaby, BC, Canada.
Gilham
D
D
Cornell
R B
RB
D000818:Animals; D001665:Binding Sites; D019556:COS Cells; D019916:Choline-Phosphate Cytidylyltransferase / Q000235:genetics / Q000378:metabolism*; D003570:Cytidine Triphosphate / Q000378:metabolism*; D004795:Enzyme Stability; D006639:Histidine; D006860:Hydrogen Bonding; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011487:Protein Conformation; D011489:Protein Denaturation; D051381:Rats; D014162:Transfection
D006639:Histidine; D003570:Cytidine Triphosphate; D019916:Choline-Phosphate Cytidylyltransferase