Ueno Y, Hata S, Izui K
Division of Applied Biosciences, Graduate School of Agriculture, Kyoto University, Japan.
FEBS Lett. 1997 Nov 3;417(1):57-60. doi: 10.1016/s0014-5793(97)01254-4.
In order to mimic regulatory phosphorylation of the Ser-15 of maize C4-form phosphoenolpyruvate carboxylase (PEPC), we replaced Ser-15 and Lys-12 with Asp (S15D) and Asn (K12N), respectively, by site-directed mutagenesis. Although both mutant enzymes were catalytically as active as the wild-type PEPC, they showed much less sensitivity to malate, an allosteric inhibitor, similarly to the phosphorylated wild-type PEPC. A maize protein kinase of 30 kDa which is known to be specific to PEPC (PEPC-PK), phosphorylated K12N as well as the wild-type PEPC but not S15D. The phosphorylation of K12N further diminished the sensitivity to malate. Thus, a positive charge of the conserved Lys-12 is not required for the recognition by PEPC-PK but contributes to the intrinsic sensitivity to malate inhibition.
为了模拟玉米C4型磷酸烯醇式丙酮酸羧化酶（PEPC）丝氨酸15位点的调节性磷酸化，我们通过定点诱变分别用天冬氨酸（S15D）和天冬酰胺（K12N）取代了丝氨酸15和赖氨酸12。尽管两种突变酶的催化活性与野生型PEPC相同，但它们对变构抑制剂苹果酸的敏感性远低于野生型PEPC，类似于磷酸化的野生型PEPC。一种已知对PEPC具有特异性的30 kDa玉米蛋白激酶（PEPC-PK），可磷酸化K12N以及野生型PEPC，但不能磷酸化S15D。K12N的磷酸化进一步降低了对苹果酸的敏感性。因此，保守的赖氨酸12的正电荷对于PEPC-PK的识别不是必需的，但有助于对苹果酸抑制的内在敏感性。
