The Structural Basis of Enzyme Catalysis: Mutagenesis of β-lactamase at Ala 172, Glu 166, and Ala 237

Citation

Healey, William Joseph (1989) The Structural Basis of Enzyme Catalysis: Mutagenesis of β-lactamase at Ala 172, Glu 166, and Ala 237. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/t2g7-x074. https://resolver.caltech.edu/CaltechETD:etd-05212007-133513

Abstract

RTEM-1 β-lactamase is one of a group of enzymes that confer resistance to penam and cephem antibiotics by hydrolizing the β-lactam bond. In order to elucidate the structural basis for its catalytics, site saturation mutagenesis was performed on alanine 172, glutamate 166, and alanine 237. Site saturation is the replacement of any codon with a mixture of codons to produce all possible mutations at a single location.

Alanine 172 was found to have no effect on penam hydrolysis, but seven mutants -- Arg, Asp, Glu, Gln, Ile, Leu, and Lys -- were inactive on cephalothin. Although no specific structural role is hypothesized for this residue, some mutations (including those that change the charge) are able to affect activity. This altered activity is most likely due to propagated structural changes.

Glutamate 166 was chosen for mutagenesis because of its probable role as a general base in the deacylation step of the catalytic mechanism. Only three mutants (Asp, His, and Tyr) exhibited low activity. The K_M was measured for several mutants and found to be the same as wild-type (20 µM), indicating that mutations at this site do not affect substrate binding. Two mutants, Lys and Arg, yielded proteins that were unstable at 37°C, possibly because of an unfavorable interaction with lysine 73. A double mutant was constructed to replace Lys 73 with Glu while keeping Lys at 166; full cellular stability was restored.

Finally, the series of mutants at alanine 237 was produced; this residue is important to catalysis since it makes up part of the oxyanion hole that stabilizes a catalytic intermediate. Of all mutants at this site, only proline was inactive. Two mutants showed increased activity on cephems, asparagine (which had 380% of wild-type activity) and threonine (150%), revealing that this site is involved in substrate specificity.

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