Veitch D P, Cornell R B
Institute of Molecular Biology and Biochemistry, Simon Fraser University, Burnaby, British Columbia, Canada.
Biochemistry. 1996 Aug 20;35(33):10743-50. doi: 10.1021/bi960402c.
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.
通过从双突变体CT-S91C114构建单突变体CT-S91和CT-C114，研究了CTP：磷酸胆碱胞苷转移酶假定催化结构域中的突变效应，先前已证明该双突变体的活性比野生型低4倍[Walkey，C.R.，Kalmar，G.B.，& Cornell，R.B.（1994）J.Biol.Chem.269，5742 - 5749]。构建体在COS细胞中过表达。发现甘氨酸91突变为丝氨酸91是活性降低的原因，而丝氨酸114突变为半胱氨酸114没有影响。91位的丙氨酸替代物CT-A91对胞苷转移酶活性的影响较小。从COS细胞中纯化CT-S91和CT-WT，并测定它们的动力学常数。CT-S91的Vmax低4倍，CTP的K(m)比野生型酶高25倍，这表明用丝氨酸替代甘氨酸91会干扰CTP结合。CT-S91突变体中磷酸胆碱的K(m)不受影响。CT-S91和CT-WT对胰凝乳蛋白酶的敏感性没有差异，这表明该突变没有引起蛋白质结构的全局性变化。然而，CT-S91的活性比CT-WT更容易受到变性剂尿素的抑制，这表明活性位点折叠受到了干扰。甘氨酸91位于局部序列HSGH中，该序列在新型胞苷转移酶超家族中被认为是一个CTP结合基序[Bork，P.，Holm，L.，Koonin，E.V.，& Sander，C.（1995）Proteins：Struct.，Funct.，Genet.22，259 - 266]。我们的结果是对这一假设的首次实验验证。
