Janiak-Spens Fabiola, Cook Paul F, West Ann H
Department of Chemistry and Biochemistry, University of Oklahoma, Norman, Oklahoma 73019, USA.
Biochemistry. 2005 Jan 11;44(1):377-86. doi: 10.1021/bi048433s.
In Saccharomyces cerevisiae, the histidine-containing phosphotransfer (HPt) protein YPD1 transfers phosphoryl groups between the three different response regulator domains of SLN1, SSK1, and SKN7 (designated R1, R2, and R3, respectively). Together these proteins form a branched histidine-aspartic acid phosphorelay system through which cells can respond to hyperosmotic and other environmental stresses. The in vivo order of phosphotransfer reactions is believed to proceed from SLN1-R1 to YPD1 and then subsequently to SSK1-R2 or SKN7-R3. The individual phosphoryl transfer reactions between YPD1 and the response regulator domains have been examined kinetically. A maximum forward rate constant of 29 s(-)(1) was determined for the reaction between SLN1-R1 approximately P and YPD1 with a K(d) of 1.4 microM for the SLN1-R1 approximately P.YPD1 complex. In the subsequent reactions, phosphotransfer from YPD1 to SSK1-R2 is very rapid (160 s(-)(1)) and is strongly favored over phosphotransfer to SKN7-R3. Phosphotransfer reactions between YPD1 and SLN1-R1 or SKN7-R3 were reversible. In contrast, no reverse transfer from SSK1-R2 approximately P to YPD1 was observed. These findings are consistent with the notion that SSK1 is constitutively phosphorylated under normal osmotic conditions. In addition, we have examined the roles of several conserved amino acid residues surrounding the phosphorylatable histidine (H64) of YPD1 using phosphoryl transfer reactions involving YPD1 mutants. With respect to phosphoryl transfer from SLN1-R1 approximately P, only one YPD1 mutant (K67A) exhibited an increase in K(d) and thus affects binding of YPD1 to SLN1-R1 approximately P, whereas other mutants (R90A, Q86A, and G68Q) showed a decrease in phosphoryl transfer rate. Only the G68Q-YPD1 mutant was significantly affected in phosphotransfer to SSK1-R2 ( approximately 680-fold decrease in rate in comparison to wild-type). This is the first report of a kinetic analysis of a eukaryotic "two-component" histidine-aspartic acid phosphotransfer system, enabling a comparison of the transfer rates and binding constants to the few bacterial systems that have been studied this way.
在酿酒酵母中，含组氨酸的磷酸转移（HPt）蛋白YPD1在SLN1、SSK1和SKN7的三个不同应答调节结构域（分别命名为R1、R2和R3）之间转移磷酸基团。这些蛋白共同形成一个分支的组氨酸 - 天冬氨酸磷酸中继系统，细胞通过该系统可对高渗和其他环境胁迫作出反应。磷酸转移反应的体内顺序被认为是从SLN1 - R1到YPD1，然后再到SSK1 - R2或SKN7 - R3。已对YPD1与应答调节结构域之间的单个磷酸转移反应进行了动力学研究。确定SLN1 - R1P与YPD1反应的最大正向速率常数为29 s⁻¹，SLN1 - R1P·YPD1复合物的解离常数Kd为1.4 μM。在随后的反应中，从YPD1到SSK1 - R2的磷酸转移非常迅速（160 s⁻¹），并且比向SKN7 - R3的磷酸转移更受青睐。YPD1与SLN1 - R1或SKN7 - R3之间的磷酸转移反应是可逆的。相比之下，未观察到从SSK1 - R2P到YPD1的逆向转移。这些发现与SSK1在正常渗透条件下组成型磷酸化的观点一致。此外，我们使用涉及YPD1突变体的磷酸转移反应研究了YPD1可磷酸化组氨酸（H64）周围几个保守氨基酸残基的作用。关于从SLN1 - R1P的磷酸转移，只有一个YPD1突变体（K67A）的Kd增加，因此影响YPD1与SLN1 - R1~P的结合，而其他突变体（R90A、Q86A和G68Q）的磷酸转移速率降低。只有G68Q - YPD1突变体在向SSK1 - R2的磷酸转移中受到显著影响（与野生型相比速率降低约680倍）。这是关于真核“双组分”组氨酸 - 天冬氨酸磷酸转移系统动力学分析的首次报道，使得能够将转移速率和结合常数与少数已以这种方式研究的细菌系统进行比较。
