Properties of a LacY efflux mutant
1Department of Physiology, University of California Los Angeles, Los Angeles, California 90095-1662, USA.
Biochemistry
|September 2, 2009
Summary
A mutation in lactose permease (LacY) enhances sugar efflux by disrupting proton binding. This finding offers insights into the transport mechanism of this crucial bacterial protein.
Area of Science:
- Biochemistry
- Structural Biology
- Membrane Transport
Background:
- Lactose permease (LacY) from Escherichia coli facilitates lactose transport via an alternating access mechanism.
- Crystal structures reveal a two-six-helix bundle with a central cavity, opening alternately to cytoplasmic and periplasmic sides.
- Key residues within this cavity are critical for sugar and proton (H+) binding.
Purpose of the Study:
- To investigate the functional consequences of a specific mutation in LacY affecting sugar transport.
- To elucidate the role of Asp240 in the LacY transport mechanism and its interaction with Lys319.
- To understand how altering the salt-bridge/H-bond network impacts LacY activity.
Main Methods:
- Site-directed mutagenesis to create an Asp240Glu mutant of LacY.
- Assays measuring active lactose transport and downhill lactose/H+ efflux.
- Analysis of sugar concentration-dependent transport kinetics.
Main Results:
- The Asp240Glu mutant showed significantly reduced active lactose transport.
- An increased rate of downhill lactose/H+ efflux was observed in the mutant.
- The mutation led to a decrease in sugar affinity and suggested a reduced affinity for H+.
Conclusions:
- The Asp240Glu mutation primarily impairs H+ binding affinity, leading to increased sugar efflux.
- This highlights the critical role of the Asp240-Lys319 interaction in symport efficiency.
- The study provides further evidence for the alternating access model of LacY function.
