Improved Energy Coupling of Human P-glycoprotein by the Glycine 185 to Valine Mutation

Hiroshi Omote, Robert A. Figler, Mark K. Polar, Marwan K. Al-Shawi

Research output: Contribution to journalArticle

33 Citations (Scopus)

Abstract

A glycine 185 to valine mutation of human P-glycoprotein (ABCB1, MDR1) has been previously isolated from high colchicine resistance cell lines. We have employed purified and reconstituted P-glycoproteins expressed in Saccharomyces cerevisiae [Figler et al. (2000) Arch. Biochem. Biophys. 376, 34-46] and devised a set of thermodynamic analyses to reveal the mechanism of improved resistance. Purified G185V enzyme shows altered basal ATPase activity but a strong stimulation of colchicine- and etoposide-dependent activities, suggesting a tight regulation of ATPase activity by these drugs. The mutant enzyme has a higher apparent Km for colchicine and a lower K m for etoposide than that of wild type. Kinetic constants for other transported drugs were not significantly modified by this mutation. Systematic thermodynamic analyses indicate that the G185V enzyme has modified thermodynamic properties of colchicine- and etoposide-dependent activities. To improve the rate of colchicine or etoposide transport, the G185V enzyme has lowered the Arrhenius activation energy of the transport rate-limiting step. The high transition state energies of wild-type P-glycoprotein, when transporting etoposide or colchicine, increase the probability of nonproductive degradation of the transition state without transport. G185V P-glycoprotein transports etoposide or colchicine in an energetically more efficient way with decreased enthalpic and entropic components of the activation energy. Our new data fully reconcile the apparently conflicting results of previous studies. EPR analysis of the spin-labeled G185C enzyme in a cysteine-less background and kinetic parameters of the G185C enzyme indicate that position 185 is surrounded by other residues and is volume sensitive. These results and atomic detail structural modeling suggest that residue 185 is a pivotal point in transmitting conformational changes between the catalytic sites and the colchicine drug binding domain. Replacement of this residue with a bulky valine alters this communication and results in more efficient transport of etoposide or colchicine.

Original languageEnglish
Pages (from-to)3917-3928
Number of pages12
JournalBiochemistry
Volume43
Issue number13
DOIs
Publication statusPublished - Apr 6 2004
Externally publishedYes

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Colchicine
Valine
P-Glycoprotein
Glycine
Etoposide
Mutation
Enzymes
Thermodynamics
Adenosine Triphosphatases
Activation energy
Pharmaceutical Preparations
P-Glycoproteins
Arches
Electron transitions
Kinetic parameters
Yeast
Electron energy levels
Cysteine
Paramagnetic resonance
Saccharomyces cerevisiae

ASJC Scopus subject areas

  • Biochemistry

Cite this

Improved Energy Coupling of Human P-glycoprotein by the Glycine 185 to Valine Mutation. / Omote, Hiroshi; Figler, Robert A.; Polar, Mark K.; Al-Shawi, Marwan K.

In: Biochemistry, Vol. 43, No. 13, 06.04.2004, p. 3917-3928.

Research output: Contribution to journalArticle

Omote, Hiroshi ; Figler, Robert A. ; Polar, Mark K. ; Al-Shawi, Marwan K. / Improved Energy Coupling of Human P-glycoprotein by the Glycine 185 to Valine Mutation. In: Biochemistry. 2004 ; Vol. 43, No. 13. pp. 3917-3928.
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