Familial mutations and zinc stoichiometry determine the rate-limiting step of nitrocefin hydrolysis by metallo-beta-lactamase from Bacteroides fragilis.
The diverse members of the metallo-beta-lactamase family are a growing clinical threat evolving under considerable selective pressure. The enzyme from Bacillus cereus differs from the Bacteroides fragilis enzyme in sequence, zinc stoichiometry, and mechanism. To chart the evolution of the more reactive B. fragilis enzyme, we have made changes in an active site cysteine residue as well as in zinc content to mimic that which occurs in the B. cereus enzyme. Specifically, by introducing a C104R mutation into the B. fragilis enzyme, binding of two zinc ions is maintained, but the k(cat) value for nitrocefin hydrolysis is decreased from 226 to 14 s(-)(1). Removal of 1 equiv of zinc from this mutant further decreases k(cat) to 4.4 s(-)(1). In both cases, the observed k(cat) closely approximates that found in the di- and monozinc forms of the B. cereus enzyme (12 and 6 s(-)(1), respectively). Pre-steady-state stopped-flow studies using nitrocefin as a substrate indicate that these enzyme forms share a similar mechanism featuring an anionic intermediate but that the rate-limiting step changes from protonation of that species to the C-N bond cleavage leading to the intermediate. Overall, features that contribute 3.7 kcal/mol toward the acceleration of the C-N bond cleavage step have been uncovered although some of the total acceleration is masked in the steady-state by a change in rate-limiting step. These experiments illustrate one step in the evolution of a catalytic mechanism and, in a larger perspective, one step in the evolution of antibiotic resistance mechanisms.
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The Pennsylvania State University, Department of Chemistry, 414 Wartik Laboratory, University Park, PA 16802, USA.
Wang
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Benkovic
S J
SJ
D019943:Amino Acid Substitution / Q000235:genetics; D001120:Arginine / Q000235:genetics; D001409:Bacillus cereus / Q000201:enzymology / Q000235:genetics; D001441:Bacteroides fragilis / Q000201:enzymology* / Q000235:genetics; D002384:Catalysis; D002511:Cephalosporins / Q000378:metabolism*; D003035:Cobalt / Q000378:metabolism; D003545:Cysteine / Q000235:genetics; D003956:Dialysis; D006868:Hydrolysis; D007202:Indicators and Reagents; D007700:Kinetics; D008667:Metalloproteins / Q000235:genetics* / Q000378:metabolism; D005810:Multigene Family / Q000235:genetics; D016297:Mutagenesis, Site-Directed; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000138:chemical synthesis / Q000302:isolation & purification / Q000378:metabolism; D012118:Resorcinols / Q000378:metabolism; D013054:Spectrophotometry, Atomic; D015032:Zinc / Q000737:chemistry* / Q000378:metabolism; D001618:beta-Lactamases / Q000235:genetics* / Q000378:metabolism
D002511:Cephalosporins; D007202:Indicators and Reagents; D008667:Metalloproteins; D011994:Recombinant Proteins; D012118:Resorcinols; D003035:Cobalt; D001120:Arginine; D001618:beta-Lactamases; C021720:nitrocefin; D015032:Zinc; D003545:Cysteine; C028398:4-(2-pyridylazo)resorcinol