18O-exchange evidence that mutations of arginine in a signature sequence for P-type pumps affect inorganic phosphate binding

Robert A. Farley, Emad - Elquza, Jochen Müller-Ehmsen, David J. Kane, Agnes K. Nagy, Vladimir N. Kasho, Larry D. Faller

Research output: Contribution to journalArticle

10 Citations (Scopus)

Abstract

We have proposed a model for part of the catalytic site of P-type pumps in which arginine in a signature sequence functions like lysine in P-loop-containing enzymes that catalyze adenosine 5′-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 DPPR sequence of Na,K-ATPase binds Mg2+ [Farley, R. A., et al. (1997) Biochemistry 36, 941-951]. It was developed by assuming that the catalytic domain of P-type pumps evolved from enzymes that catalyze phosphoryl group transfer. The functions of the positively charged amino group in P-loops are to bind substrate and to facilitate nucleophilic attack upon phosphorus by polarizing the γ-phosphorus - oxygen bond. To test the prediction that the positively charged guanidinium group of R596 in human α1 Na,K-ATPase participates in phosphoryl group transfer, the charge was progressively decreased by site-directed mutagenesis. Mutants R596K, -Q, -T, -M, -A, -G, and -E were expressed in yeast membranes, and their ability 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 negative charge (R596E) resulted in complete loss of activity. The remaining mutants with uncharged side chains had both lowered affinity for inorganic phosphate and altered phosphate isotopomer distributions, consistent with increased phosphate-off rate constants compared to that of the wild type. Therefore, mutations of R596 strengthen our hypothesis that the oppositely charged side chains of the DPPR peptide in Na,K-ATPase form a quaternary complex with magnesium phosphate.

Original languageEnglish (US)
Pages (from-to)6361-6370
Number of pages10
JournalBiochemistry
Volume40
Issue number21
DOIs
StatePublished - May 29 2001
Externally publishedYes

Fingerprint

Arginine
Phosphates
Pumps
Mutagenesis
Mutation
Site-Directed Mutagenesis
Phosphorus
Adenosine Triphosphatases
Catalytic Domain
Ca(2+) Mg(2+)-ATPase
Biochemistry
Phosphorylation
Guanidine
Enzymes
Aspartic Acid
Yeast
Adenosine
Lysine
Hydrolysis
Rate constants

ASJC Scopus subject areas

  • Biochemistry

Cite this

Farley, R. A., Elquza, E. ., Müller-Ehmsen, J., Kane, D. J., Nagy, A. K., Kasho, V. N., & Faller, L. D. (2001). 18O-exchange evidence that mutations of arginine in a signature sequence for P-type pumps affect inorganic phosphate binding. Biochemistry, 40(21), 6361-6370. https://doi.org/10.1021/bi010270+

18O-exchange evidence that mutations of arginine in a signature sequence for P-type pumps affect inorganic phosphate binding. / Farley, Robert A.; Elquza, Emad -; Müller-Ehmsen, Jochen; Kane, David J.; Nagy, Agnes K.; Kasho, Vladimir N.; Faller, Larry D.

In: Biochemistry, Vol. 40, No. 21, 29.05.2001, p. 6361-6370.

Research output: Contribution to journalArticle

Farley, Robert A. ; Elquza, Emad - ; Müller-Ehmsen, Jochen ; Kane, David J. ; Nagy, Agnes K. ; Kasho, Vladimir N. ; Faller, Larry D. / 18O-exchange evidence that mutations of arginine in a signature sequence for P-type pumps affect inorganic phosphate binding. In: Biochemistry. 2001 ; Vol. 40, No. 21. pp. 6361-6370.
@article{84354421a7414c0bbc9b381740de3b12,
title = "18O-exchange evidence that mutations of arginine in a signature sequence for P-type pumps affect inorganic phosphate binding",
abstract = "We have proposed a model for part of the catalytic site of P-type pumps in which arginine in a signature sequence functions like lysine in P-loop-containing enzymes that catalyze adenosine 5′-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 DPPR sequence of Na,K-ATPase binds Mg2+ [Farley, R. A., et al. (1997) Biochemistry 36, 941-951]. It was developed by assuming that the catalytic domain of P-type pumps evolved from enzymes that catalyze phosphoryl group transfer. The functions of the positively charged amino group in P-loops are to bind substrate and to facilitate nucleophilic attack upon phosphorus by polarizing the γ-phosphorus - oxygen bond. To test the prediction that the positively charged guanidinium group of R596 in human α1 Na,K-ATPase participates in phosphoryl group transfer, the charge was progressively decreased by site-directed mutagenesis. Mutants R596K, -Q, -T, -M, -A, -G, and -E were expressed in yeast membranes, and their ability 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 negative charge (R596E) resulted in complete loss of activity. The remaining mutants with uncharged side chains had both lowered affinity for inorganic phosphate and altered phosphate isotopomer distributions, consistent with increased phosphate-off rate constants compared to that of the wild type. Therefore, mutations of R596 strengthen our hypothesis that the oppositely charged side chains of the DPPR peptide in Na,K-ATPase form a quaternary complex with magnesium phosphate.",
author = "Farley, {Robert A.} and Elquza, {Emad -} and Jochen M{\"u}ller-Ehmsen and Kane, {David J.} and Nagy, {Agnes K.} and Kasho, {Vladimir N.} and Faller, {Larry D.}",
year = "2001",
month = "5",
day = "29",
doi = "10.1021/bi010270+",
language = "English (US)",
volume = "40",
pages = "6361--6370",
journal = "Biochemistry",
issn = "0006-2960",
publisher = "American Chemical Society",
number = "21",

}

TY - JOUR

T1 - 18O-exchange evidence that mutations of arginine in a signature sequence for P-type pumps affect inorganic phosphate binding

AU - Farley, Robert A.

AU - Elquza, Emad -

AU - Müller-Ehmsen, Jochen

AU - Kane, David J.

AU - Nagy, Agnes K.

AU - Kasho, Vladimir N.

AU - Faller, Larry D.

PY - 2001/5/29

Y1 - 2001/5/29

N2 - We have proposed a model for part of the catalytic site of P-type pumps in which arginine in a signature sequence functions like lysine in P-loop-containing enzymes that catalyze adenosine 5′-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 DPPR sequence of Na,K-ATPase binds Mg2+ [Farley, R. A., et al. (1997) Biochemistry 36, 941-951]. It was developed by assuming that the catalytic domain of P-type pumps evolved from enzymes that catalyze phosphoryl group transfer. The functions of the positively charged amino group in P-loops are to bind substrate and to facilitate nucleophilic attack upon phosphorus by polarizing the γ-phosphorus - oxygen bond. To test the prediction that the positively charged guanidinium group of R596 in human α1 Na,K-ATPase participates in phosphoryl group transfer, the charge was progressively decreased by site-directed mutagenesis. Mutants R596K, -Q, -T, -M, -A, -G, and -E were expressed in yeast membranes, and their ability 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 negative charge (R596E) resulted in complete loss of activity. The remaining mutants with uncharged side chains had both lowered affinity for inorganic phosphate and altered phosphate isotopomer distributions, consistent with increased phosphate-off rate constants compared to that of the wild type. Therefore, mutations of R596 strengthen our hypothesis that the oppositely charged side chains of the DPPR peptide in Na,K-ATPase form a quaternary complex with magnesium phosphate.

AB - We have proposed a model for part of the catalytic site of P-type pumps in which arginine in a signature sequence functions like lysine in P-loop-containing enzymes that catalyze adenosine 5′-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 DPPR sequence of Na,K-ATPase binds Mg2+ [Farley, R. A., et al. (1997) Biochemistry 36, 941-951]. It was developed by assuming that the catalytic domain of P-type pumps evolved from enzymes that catalyze phosphoryl group transfer. The functions of the positively charged amino group in P-loops are to bind substrate and to facilitate nucleophilic attack upon phosphorus by polarizing the γ-phosphorus - oxygen bond. To test the prediction that the positively charged guanidinium group of R596 in human α1 Na,K-ATPase participates in phosphoryl group transfer, the charge was progressively decreased by site-directed mutagenesis. Mutants R596K, -Q, -T, -M, -A, -G, and -E were expressed in yeast membranes, and their ability 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 negative charge (R596E) resulted in complete loss of activity. The remaining mutants with uncharged side chains had both lowered affinity for inorganic phosphate and altered phosphate isotopomer distributions, consistent with increased phosphate-off rate constants compared to that of the wild type. Therefore, mutations of R596 strengthen our hypothesis that the oppositely charged side chains of the DPPR peptide in Na,K-ATPase form a quaternary complex with magnesium phosphate.

UR - http://www.scopus.com/inward/record.url?scp=0035967445&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0035967445&partnerID=8YFLogxK

U2 - 10.1021/bi010270+

DO - 10.1021/bi010270+

M3 - Article

C2 - 11371198

AN - SCOPUS:0035967445

VL - 40

SP - 6361

EP - 6370

JO - Biochemistry

JF - Biochemistry

SN - 0006-2960

IS - 21

ER -