The Variation of Catalytic Efficiency of Bacillus cereus Metallo-β-lactamase with Different Active Site Metal Ions

Adriana Badarau, Michael I. Page

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48 Citations (Scopus)


The kinetics and mechanism of hydrolysis of the native zinc and metal substituted Bacillus cereus (BcII) metallo-β-lactamase have been investigated. The pH and metal ion dependence of kcat and k cat/Km, determined under steady-state conditions, for the cobalt substituted BcII catalyzed hydrolysis of cefoxitin, cephaloridine, and cephalexin indicate that an enzyme residue of apparent pKa 6.3 ± 0.1 is required in its deprotonated form for metal ion binding and catalysis. The kcat/Km for cefoxitin and cephalexin with cadmium substituted BcII is dependent on two ionizing groups on the enzyme: one of pKa1 = 8.7 ± 0.1 required in its deprotonated form and the other of pKa2 = 9.3 ± 0.1 required in its protonated form for activity. The pH dependence of the competitive inhibition constant, K i, for CdBcII with L-captopril indicates that pKa1 = 8.7 ± 0.1 corresponds to the cadmium-bound water. For the manganese substituted BcII, the pH dependence of kcat/Km for benzylpenicillin, cephalexin, and cefoxitin similarly indicated the importance of two catalytic groups: one of pKa1 = 8.5 ± 0.1 which needs to be deprotonated and the other of pKa2 = 9.4 ± 0.1 which needs to be protonated for catalysis; the pKa1 was assigned to the manganese-bound water. The rate was metal ion concentration dependent at the highest manganese concentrations used (10-3 M). The metal substituted species have similar or higher catalytic activities compared with the zinc enzyme, albeit at pHs above 7. Interestingly, with cefoxitin, a very poor substrate for ZnBcII, both kcat and kcat/Km increase with increasing pKa of the metal-bound water, in the order Zn < Co < Mn < Cd. A higher pKa for the metal-bound water for cadmium and manganese BCII leads to more reactive enzymes than the native zinc BcII, suggesting that the role of the metal ion is predominantly to provide the nucleophilic hydroxide, rather than to act as a Lewis acid to polarize the carbonyl group and stabilize the oxyanion tetrahedral intermediate.

Original languageEnglish
Pages (from-to)10654-10666
Number of pages13
Issue number35
Early online date15 Aug 2006
Publication statusPublished - 5 Sep 2006


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