Structural determinants of substrate binding to Bacillus cereus metallo-beta-lactamase.
Binding and hydrolysis of the beta-lactams cefotaxime, cephapirin, imipenem, and benzylpenicillin by the metallo-beta-lactamase from Bacillus cereus were studied by presteady state kinetic measurements. In all cases, the substrate was unmodified in the most populated reaction intermediate, and no chemically modified substrate species accumulated to a detectable amount. The cephalosporins tested showed similar formation rate constants for this intermediate, and they differed mostly in their decay rates. Formation of a non-productive enzyme.substrate complex was detected for imipenem. The substrate binding differences can be accounted for by considering the structural features of each substrate. The apoenzyme could not bind any of the substrates, but binding was restored when the apoenzyme was reconstituted with Zn(II), revealing that the metal ions are the main determinants of substrate binding. This evidence is in line with the lack of an optimized substrate recognition patch in B1 and B3 metallo-beta-lactamases that provides a broad substrate spectrum.
Rasia
Rodolfo M
RM
Area Biofisica and Instituto de Biologia Molecular y Celular de Rosario, Facultad de Ciencias Bioquimicas y Farmaceuticas, Universidad Nacional de Rosario, Suipacha 531, S2002LRK, Rosario, Argentina.
Vila
Alejandro J
AJ
D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D001409:Bacillus cereus / Q000201:enzymology*; D002439:Cefotaxime / Q000494:pharmacology; D002514:Cephapirin / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D006868:Hydrolysis; D015378:Imipenem / Q000494:pharmacology; D007477:Ions; D007700:Kinetics; D008956:Models, Chemical; D010400:Penicillin G / Q000494:pharmacology; D011485:Protein Binding; D011487:Protein Conformation; D013379:Substrate Specificity; D013696:Temperature; D013997:Time Factors; D015032:Zinc / Q000737:chemistry; D001618:beta-Lactamases / Q000737:chemistry*
D000900:Anti-Bacterial Agents; D007477:Ions; D015378:Imipenem; D002514:Cephapirin; D001618:beta-Lactamases; D015032:Zinc; D002439:Cefotaxime; D010400:Penicillin G