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beta-Lactamase mutations far from the active site influence inhibitor binding
R A Bonomo1, C G Dawes, J R Knox
1Research Service, Department of Veterans Affairs Medical Center, Cleveland, OH.
Biochimica Et Biophysica Acta
|February 22, 1995
Summary
A specific mutation in beta-lactamase enzymes impacts clavulanic acid resistance by altering a key hydrogen bond interaction. This finding highlights the role of residue 276 in enzyme structure and function.
Area of Science:
- Biochemistry
- Molecular Biology
- Enzymology
Background:
- Class A beta-lactamases are crucial enzymes in bacterial resistance.
- Arg-244 is a conserved residue vital for inactivation by clavulanic acid.
- This residue's positioning is maintained by a hydrogen bond from residue 276.
Purpose of the Study:
- To investigate the importance of the hydrogen bond interaction involving residue 276.
- To analyze the effect of an Asn276-Gly mutation on beta-lactamase activity and inhibitor susceptibility.
Main Methods:
- Construction of an Asn276-Gly mutant of the OHIO-1 beta-lactamase.
- Determination of minimum inhibitory concentrations (MICs) against various beta-lactams and inhibitors.
- Enzyme kinetic analysis to determine inhibition constants (Ki) and catalytic efficiency (Vmax/Km).
Main Results:
- The Asn276-Gly mutant showed increased resistance to clavulanate but similar susceptibility to sulbactam and tazobactam.
- Apparent Ki for clavulanate was 10-fold higher in the mutant, while Ki for sulbactam and tazobactam decreased.
- Mutant enzyme exhibited reduced affinity (Km) and catalytic efficiency (Vmax/Km) for most beta-lactam substrates.
Conclusions:
- The hydrogen bond from residue 276 is critical for correctly orienting Arg-244 in the active site.
- Alterations in this interaction significantly affect the enzyme's response to specific beta-lactamase inhibitors.
- This study emphasizes the role of specific hydrogen bonding networks in beta-lactamase structure-function relationships.