The role of tyrosine 150 in catalysis of beta-lactam hydrolysis by AmpC beta-lactamase from Escherichia coli investigated by site-directed mutagenesis.
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.
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Department of Molecular Microbiology, Washington University School of Medicine, St. Louis, Missouri 63110-1093.
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D000215:Acylation; D000900:Anti-Bacterial Agents / Q000378:metabolism*; D001398:Aztreonam / Q000378:metabolism; D001426:Bacterial Proteins; D002384:Catalysis; D002443:Ceftriaxone / Q000378:metabolism; D002512:Cephalothin / Q000378:metabolism; D002942:Circular Dichroism; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D006868:Hydrolysis; D007700:Kinetics; D008712:Methicillin / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D010957:Plasmids; D017433:Protein Structure, Secondary; D015183:Restriction Mapping; D013329:Structure-Activity Relationship; D014443:Tyrosine / Q000737:chemistry / Q000235:genetics; D001618:beta-Lactamases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D014443:Tyrosine; D002443:Ceftriaxone; C048795:AmpC beta-lactamases; D001618:beta-Lactamases; D001398:Aztreonam; D008712:Methicillin; D002512:Cephalothin