Single amino acid substitution in the putative transmembrane helix V in KdpB of the KdpFABC complex of Escherichia coli uncouples ATPase activity and ion transport

Autor(en): Bramkamp, M
Altendorf, K 
Stichwörter: Biochemistry & Molecular Biology; CA2+-ATPASE; CALCIUM-PUMP; CHANNELS; CRYSTAL-STRUCTURE; ENERGY TRANSDUCTION; INDUCED CONFORMATIONAL-CHANGES; K+-BINDING; SARCOPLASMIC-RETICULUM; SELECTIVITY FILTER; SUBUNIT
Erscheinungsdatum: 2005
Herausgeber: AMER CHEMICAL SOC
Journal: BIOCHEMISTRY
Volumen: 44
Ausgabe: 23
Startseite: 8260
Seitenende: 8266
Zusammenfassung: 
The KdpFABC complex, found in a variety of prokaryotes, is an emergency potassium uptake system which belongs to the family of P-type ATPases. Site-directed mutagenesis of the charged residues aspartate 583 and lysine 586 in the putative transmembrane helix V Of Subunit KdpB revealed that these charges are involved in the coupling of ATP hydrolysis and ion translocation. Phenotypic characterization of KdpFABC derivatives carrying alterations at either D583 or K586 demonstrated that only restoration of charges at these positions allowed growth on low potassium concentrations. Substitutions. which eliminated the negative charge at position 583, did not allow growth below 15 mM potassium on solid media. In contrast, substitutions of the positive charge at position 586 allowed growth down to 0.3 mM potassium. Purified KdpFABC complexes carrying these substitutions exhibited ATPase activity. which was, however, found to be comparatively resistant to o-vanadate. Furthermore, elimination of the charges led to a complete loss of ion-stimulated ATPase activity, though the rate of hydrolysis was comparable to wild-type activity, indicating an uncoupling between ATP hydrolysis and ion translocation, This fact was substantiated by reconstitution experiments, in which the D583A complex was unable to facilitate ion translocation, whereas the D583E mutant complex still exhibited such activity. On the basis of these results, a new transport model for the Kdp-ATPase is presented here, in which the amino acids D593 and K586 are supposed to play a role in the gating mechanism of the complex. Furthermore. movement of the charged side chains could have a direct influence on the free energy profile within the potassium transporting subunit KdpA, thereby facilitating ion transport against the concentration gradient into the cytosol.
ISSN: 00062960
DOI: 10.1021/bi050135n

Show full item record

Page view(s)

1
Last Week
0
Last month
1
checked on Mar 4, 2024

Google ScholarTM

Check

Altmetric