Concerted Conformational Effects of Ca2+ and ATP Are Required for Activation of Sequential Reactions in the Ca2+ ATPase (SERCA) Catalytic Cycle
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- 作者：Giuseppe Inesi ; David Lewis ; Hailun Ma ; Anand Prasad ; Chikashi Toyoshima
- 刊名：Biochemistry
- 出版年：2006
- 出版时间：November 21, 2006
- 年：2006
- 卷：45
- 期：46
- 页码：13769 - 13778
- 全文大小：814K
- 年卷期：v.45,no.46(November 21, 2006)
- ISSN：1520-4995
文摘
We relate solution behavior to the crystal structure of the Ca2+ ATPase (SERCA). We findthat nucleotide binding occurs with high affinity through interaction of the adenosine moiety with the Ndomain, even in the absence of Ca2+ and Mg2+, or to the closed conformation stabilized by thapsigargin(TG). Why then is Ca2+ crucial for ATP utilization? The influence of adenosine 5'-(
,
-methylene)triphosphate (AMPPCP), Ca2+, and Mg2+ on proteolytic digestion patterns, interpreted in the light ofknown crystal structures, indicates that a Ca2+-dependent conformation of the ATPase headpiece is requiredfor a further transition induced by nucleotide binding. This includes opening of the headpiece, which inturn allows inclination of the "A" domain and bending of the "P" domain. Thereby, the phosphate chainof bound ATP acquires an extended configuration allowing the -phosphate to reach Asp351 to form acomplex including Mg2+. We demonstrate by Asp351 mutation that this "productive" conformation ofthe substrate-enzyme complex is unstable because of electrostatic repulsion at the phosphorylation site.However, this conformation is subsequently stabilized by covalent engagement of the -phosphate yieldingthe phosphoenzyme intermediate. We also demonstrate that the ADP product remains bound with highaffinity to the transition state complex but dissociates with lower affinity as the phosphoenzyme undergoesa further conformational change (i.e., E1-P to E2-P transition). Finally, we measured low-affinity ATPbinding to stable phosphoenzyme analogues, demonstrating that the E1-P to E2-P transition and theenzyme turnover are accelerated by ATP binding to the phosphoenzyme in exchange for ADP.
,-methylene)triphosphate (AMPPCP), Ca2+, and Mg2+ on proteolytic digestion patterns, interpreted in the light ofknown crystal structures, indicates that a Ca2+-dependent conformation of the ATPase headpiece is requiredfor a further transition induced by nucleotide binding. This includes opening of the headpiece, which inturn allows inclination of the "A" domain and bending of the "P" domain. Thereby, the phosphate chainof bound ATP acquires an extended configuration allowing the -phosphate to reach Asp351 to form acomplex including Mg2+. We demonstrate by Asp351 mutation that this "productive" conformation ofthe substrate-enzyme complex is unstable because of electrostatic repulsion at the phosphorylation site.However, this conformation is subsequently stabilized by covalent engagement of the -phosphate yieldingthe phosphoenzyme intermediate. We also demonstrate that the ADP product remains bound with highaffinity to the transition state complex but dissociates with lower affinity as the phosphoenzyme undergoesa further conformational change (i.e., E1-P to E2-P transition). Finally, we measured low-affinity ATPbinding to stable phosphoenzyme analogues, demonstrating that the E1-P to E2-P transition and theenzyme turnover are accelerated by ATP binding to the phosphoenzyme in exchange for ADP.