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:phosph ocholine cytidylyltransferase was investigated by constructing the sin gle mutants CT-S91 and CT-C114 from the double mutant CT-S91C114, prev iously shown to have 4-fold lower than wild-type activity [Walkey, C. R., Kalmar, G. B., & Cornell, R. B. (1994) J. Biol. Chem. 269, 5742-57 49]. The constructs were overexpressed in COS cells. The mutation Gly- 91 to Ser-91 was found to be responsible for the decreased activity, w hereas Ser-114 to Cys-114 had no effect. An alanine substitution at po sition 91, CT-A91, had a lesser effect on cytidylyltransferase activit y. CT-S91 and CT-WT were purified from COS cells, and their kinetic co nstants were determined. CT-S91 had a 4-fold lower V-max, and a K-m fo r CTP 25-fold higher than the wild-type enzyme, suggesting that substi tution 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 dif ference in the chymotrypsin sensitivities of CT-S91 and CT-WT, indicat ing that the mutation did not cause a global change in protein structu re. 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 cytidy lyltransferase superfamily [Bork, P., Helm, L., Koonin, E. V., & Sande r, C. (1995) Proteins: Struct., Funct. Genet. 22, 259-266]. Our result s represent the first experimental validation of this hypothesis.