18O-Exchange Evidence That Mutations of Arginine in a Signature Sequence for P-Type Pumps Affect Inorganic Phosphate Binding
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- 作者：Robert A. Farley ; Emad Elquza ; Jochen Mü ; ller-Ehmsen ; David J. Kane ; Agnes K. Nagy ; Vladimir N. Kasho ; and Larry D. Faller
- 刊名：Biochemistry
- 出版年：2001
- 出版时间：May 29, 2001
- 年：2001
- 卷：40
- 期：21
- 页码：6361 - 6370
- 全文大小：175K
- 年卷期：v.40,no.21(May 29, 2001)
- ISSN：1520-4995
文摘
We have proposed a model for part of the catalytic site of P-type pumps in which arginine ina signature sequence functions like lysine in P-loop-containing enzymes that catalyze adenosine5'-triphosphate hydrolysis [Smirnova, I. N., Kasho, V. N., and Faller, L. D. (1998) FEBS Lett. 431, 309-314]. The model originated with evidence from site-directed mutagenesis that aspartic acid in the DPPRsequence of Na,K-ATPase binds Mg2+ [Farley, R. A., et al. (1997) Biochemistry 36, 941-951]. It wasdeveloped by assuming that the catalytic domain of P-type pumps evolved from enzymes that catalyzephosphoryl group transfer. The functions of the positively charged amino group in P-loops are to bindsubstrate and to facilitate nucleophilic attack upon phosphorus by polarizing the 
-phosphorus-oxygenbond. To test the prediction that the positively charged guanidinium group of R596 in human 
1 Na,K-ATPase participates in phosphoryl group transfer, the charge was progressively decreased by site-directedmutagenesis. Mutants R596K, -Q, -T, -M, -A, -G, and -E were expressed in yeast membranes, and theirability to catalyze phosphorylation with inorganic phosphate was evaluated by following 18O exchange.R596K, in which the positive charge is retained, resembled the wild type. Substitution of a negativecharge (R596E) resulted in complete loss of activity. The remaining mutants with uncharged side chainshad both lowered affinity for inorganic phosphate and altered phosphate isotopomer distributions, consistentwith increased phosphate-off rate constants compared to that of the wild type. Therefore, mutations ofR596 strengthen our hypothesis that the oppositely charged side chains of the DPPR peptide in Na,K-ATPase form a quaternary complex with magnesium phosphate.
-phosphorus-oxygenbond. To test the prediction that the positively charged guanidinium group of R596 in human 1 Na,K-ATPase participates in phosphoryl group transfer, the charge was progressively decreased by site-directedmutagenesis. Mutants R596K, -Q, -T, -M, -A, -G, and -E were expressed in yeast membranes, and theirability to catalyze phosphorylation with inorganic phosphate was evaluated by following 18O exchange.R596K, in which the positive charge is retained, resembled the wild type. Substitution of a negativecharge (R596E) resulted in complete loss of activity. The remaining mutants with uncharged side chainshad both lowered affinity for inorganic phosphate and altered phosphate isotopomer distributions, consistentwith increased phosphate-off rate constants compared to that of the wild type. Therefore, mutations ofR596 strengthen our hypothesis that the oppositely charged side chains of the DPPR peptide in Na,K-ATPase form a quaternary complex with magnesium phosphate.