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Binding of the transition state analog MgADP-fluoroaluminate to F1-ATPase
S Nadanaciva1, J Weber, A E Senior
1Department of Biochemistry and Biophysics, University of Rochester Medical Center, Rochester, New York 14642, USA.
Abstract:
Escherichia coli F1-ATPase from mutant betaY331W was potently inhibited by fluoroaluminate plus MgADP but not by MgADP alone. beta-Trp-331 fluorescence was used to measure MgADP binding to catalytic sites. Fluoroaluminate induced a very large increase in MgADP binding affinity at catalytic site one, a smaller increase at site two, and no effect at site three. Mutation of either of the critical catalytic site residues beta-Lys-155 or beta-Glu-181 to Gln abolished the effects of fluoroaluminate on MgADP binding. The results indicate that the MgADP-fluoroaluminate complex is a transition state analog and independently demonstrate that residues beta-Lys-155 and (particularly) beta-Glu-181 are important for generation and stabilization of the catalytic transition state. Dicyclohexylcarbodiimide-inhibited enzyme, with 1% residual steady-state ATPase, showed normal transition state formation as judged by fluoroaluminate-induced MgADP binding affinity changes, consistent with a proposed mechanism by which dicyclohexylcarbodiimide prevents a conformational interaction between catalytic sites but does not affect the catalytic step per se. The fluorescence technique should prove valuable for future transition state studies of F1-ATPase.
Insights
Fluoroaluminate and MgADP act as a transition state analog for Escherichia coli F1-ATPase. Specific mutations highlight the roles of beta-Lys-155 and beta-Glu-181 in stabilizing this catalytic transition state.
Area of Science:
- Biochemistry
- Enzyme kinetics
- Molecular biology
Background:
- Escherichia coli F1-ATPase is a crucial enzyme for cellular energy production.
- Understanding the catalytic mechanism and transition states of F1-ATPase is vital for enzyme research.
Purpose of the Study:
- To investigate the role of fluoroaluminate and MgADP as a transition state analog for F1-ATPase.
- To elucidate the function of specific catalytic site residues (beta-Lys-155, beta-Glu-181) in transition state stabilization.
- To assess the impact of dicyclohexylcarbodiimide inhibition on transition state formation.
Main Methods:
- Utilizing a betaY331W mutant of E. coli F1-ATPase with beta-Trp-331 fluorescence.
- Measuring MgADP binding affinity to catalytic sites in the presence of fluoroaluminate.
- Site-directed mutagenesis of key catalytic residues (beta-Lys-155, beta-Glu-181).
- Assessing transition state formation in dicyclohexylcarbodiimide-inhibited enzyme.
Main Results:
- Fluoroaluminate plus MgADP potently inhibited the mutant F1-ATPase, acting as a transition state analog.
- Fluoroaluminate significantly increased MgADP binding affinity at catalytic sites one and two, but not three.
- Mutations at beta-Lys-155 and beta-Glu-181 abolished fluoroaluminate's effect on MgADP binding.
- Dicyclohexylcarbodiimide-inhibited enzyme exhibited normal transition state formation, suggesting its inhibition targets inter-site communication.
Conclusions:
- The MgADP-fluoroaluminate complex serves as a valid transition state analog for F1-ATPase.
- Residues beta-Lys-155 and beta-Glu-181 are critical for the generation and stabilization of the catalytic transition state.
- The fluorescence-based method is a valuable tool for future F1-ATPase transition state studies.