Substitution of serine for glycine-91 in the HXGH motif of CTP:phosphocholine cytidylyltransferase implicates this motif in CTP binding.

The effect of mutations in the proposed catalytic domain of CTP:phosphocholine cytidylyltransferase was investigated by constructing the single mutants CT-S91 and CT-C114 from the double mutant CT-S91C114, previously shown to have 4-fold lower than wild-type activity [Walkey, C.R., Kalmar, G. B., & Cornell, R. B. (1994) J. Biol. Chem. 269, 5742-5749]. The constructs were overexpressed in COS cells. The mutation Gly-91 to Ser-91 was found to be responsible for the decreased activity, whereas Ser-114 to Cys-114 had no effect. An alanine substitution at position 91, CT-A91, had a lesser effect on cytidylyltransferase activity. CT-S91 and CT-WT were purified from COS cells, and their kinetic constants were determined. CT-S91 had a 4-fold lower Vmax, and a K(m) for CTP 25-fold higher than the wild-type enzyme, suggesting that substitution of Gly-91 with serine interferes with CTP binding. The K(m) for phosphocholine was not affected in the CT-S91 mutant. There was no difference in the chymotrypsin sensitivities of CT-S91 and CT-WT, indicating that the mutation did not cause a global change in protein structure. However, the CT-S91 activity was more susceptible to inhibition by the denaturant urea than that of CT-WT, indicative of a perturbation of the active site folding. Gly-91 resides in the local sequence HSGH, which has been proposed to be a CTP-binding motif in the novel cytidylyltransferase superfamily [Bork, P., Holm, L., Koonin, E.V., & Sander, C. (1995) Proteins: Struct., Funct., Genet. 22, 259-266]. Our results represent the first experimental validation of this hypothesis.