The Role of Tyrosine 150 in Catalysis of .beta.-Lactam Hydrolysis by AmpC .beta.-Lactamase from Escherichia coli Investigated by Site-Directed Mutagenesis

Abstract

The kinetics of beta-lactam hydrolysis by wild-type AmpC beta-lactamase from Escherichia coli and three mutant proteins created by substitution of tyrosine 150 have been examined. The catalytic efficiency was decreased 10- to 1000-fold according to the substrate and mutant being studied. The effect of the mutation was much stronger with rapidly hydrolyzed substrates (e.g., cephalothin) than it was with slowly hydrolyzed substrates (e.g., ceftriaxone). With the latter substrates, the mutagenesis had a much stronger effect on apparent affinity than it did on rates of catalysis. Indeed, the enzyme appeared to be more reactive toward certain of the slowly hydrolyzed substances (e.g., methicillin, aztreonam, and ceftriaxone). These observations were not compatible with an obligatory role of tyrosine 150 in catalysis. The analysis of the effects of the mutation on activity was complicated by the observation of at least two, kinetically distinct, forms of the enzymes. It appeared that mutation of tyrosine 150 influenced the kinetic properties of one state and that this residue is involved in the partitioning of the enzyme between the different reactive states.

Hydrolysis Spectrum Extension of CMY-2-Like β-Lactamases Resulting from Structural Alteration in the Y-X-N Loop

“…The biochemical alterations induced by the R148H substitution were not in agreement with a modification of the Tyr-150 residue that was previously shown to reduce the catalytic efficiency against ESCs (11). They also did not agree with structural alterations in the R2 binding site that increased affinity for ESCs and imipenem (lower K m value) (12,24,32).…”

AmpC and AmpH, proteins related to the class C beta-lactamases, bind penicillin and contribute to the normal morphology of Escherichia coli

“…Sequence similarities place AmpH among the class C ␤-lactamases, of which AmpC is also a member, but although AmpC displays marked ␤-lactamase activity as measured by hydrolysis of nitrocefin, AmpH displays no such activity toward this substrate. The observation that AmpC binds ␤-lactams has been made previously: an AmpCaztreonam complex is extremely stable, with a deacylation rate of less than 0.0001 sec Ϫ1 , and an AmpC-moxalactam complex has a deacylation rate of less than 0.001 sec Ϫ1 (13). A logical implication of their behavior as PBP-like proteins is that AmpC and/or AmpH might also play normal physiological roles in E. coli, perhaps in concert with the classic PBPs.…”

Plasmid-Determined AmpC-Type β-Lactamases

“…A Lys-Ser/ Thr-Gly motif has been found at residues 315 to 317 and plays an essential role in forming the tertiary structure of the active site. A tyrosine residue at position 150 forms part of the class C-typical motif Tyr-X-Asn and is also important (but not essential) for catalysis of ␤-lactam hydrolysis (21,64) A dendrogram of chromosomal and plasmid-encoded AmpC enzymes (Fig. 1) demonstrates the diversity of chromosomal AmpC genes and the close relationship of some plasmid-mediated enzymes to chromosomal enzymes of particular organisms.…”