Direct Evidence That the Reaction Intermediate of Metallo-β-lactamase L1 Is Metal Bound

Abstract

In an effort to probe the structure of the reaction intermediate of metallo-beta-lactamase L1 when reacted with nitrocefin and other beta-lactams, time-dependent absorption and rapid-freeze-quench (RFQ) EPR spectra were obtained using the Co(II)-substituted form of the enzyme. When using nitrocefin as the substrate, time-dependent absorption spectra demonstrate that Co(II)-substituted L1 utilizes a reaction mechanism, similar to that of the native Zn(II) enzyme, in which a short-lived intermediate forms. RFQ-EPR spectra of this intermediate demonstrate that the binding of substrate results in a change in the electronic properties of one or both of the Co(II)'s in the enzyme that is consistent with a change in the coordination sphere of this metal ion. This observation provides evidence that the reaction intermediate is a metal-bound species. RFQ-EPR studies also demonstrate that other beta-lactams, such as cephalothin, meropenem, and penicillin G, proceed through an electronically similar complex and that the role of metal is similar in all cases. EPR spectroscopy has also identified distinct product-bound species of L1, indicating that reversible product binding must be considered in all future kinetic mechanisms. Consideration of the time-dependent optical and EPR studies in light of available crystallographic information indicates the intimate involvement of the metal ion in the Zn(2)-binding site of L1 in the hydrolytic reaction.

Mechanistic Studies on the Mononuclear ZnII-Containing Metallo-β-lactamase ImiS from Aeromonas sobria

“…It is important to note that the recent computational study suggested that a tetrahedral intermediate does not form in the proposed mechanism of CphA (52), and our data cannot support nor refute this hypothesis. It is entirely possible that EI is in fact a ring-opened, unprotonated species, similar to that reported in studies with CcrA and L1 when reacted with nitrocefin (17,19,21). In either case, a proton must be transferred to the ring-opened, nitrogen to generate product.…”

“…The product signal was isolated ( Figure 7F) by subtraction of the resting signal from Figure 6I and could be simulated ( Figure 7G) assuming S = 3/2, M S = |± 1/2〉, g x,y = 2.35, g z = 2.12, E/D = 0.13. An enzyme bound product signal was also observed in RFQ EPR spectra of B3 metallo-β-lactamase L1 (21).…”

Polyadenylation factor CPSF-73 is the pre-mRNA 3'-end-processing endonuclease

“…In canonical metallo-β-lactamases, the hydroxide that bridges the two zinc ions is the nucleophile for the hydrolysis reaction, and the substrate is directly liganded to the zinc atoms 23 . In our structure, the hydroxide is placed directly below the sulfate group (Fig.…”

X-ray Absorption Spectroscopy of the Zinc-Binding Sites in the Class B2 Metallo-β-lactamase ImiS from Aeromonas veronii bv. sobria

“…Considerable information exists regarding the B1 and B3 enzymes, including X-ray diffraction (15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26)(27)(28)(29), spectroscopic (9,(30)(31)(32)(33)(34)(35)(36)(37)(38)(39)(40)(41)(42), mechanistic (43)(44)(45)(46)(47)(48)(49)(50)(51)(52)(53)(54)(55)(56)(57)(58)(59), and computational studies (60)(61)(62)(63)(64)…”