Substrate-activated Zinc Binding of Metallo-β-lactamases

Abstract

We have investigated the influence of substrate binding on the zinc ion affinity of representatives from the three metallo-␤-lactamase subclasses, B1 (BcII from Bacillus cereus and BlaB from Chryseobacterium meningosepticum), B2 (CphA from Aeromonas hydrophila), and B3 (L1 from Stenotrophomonas maltophilia). By competition experiments with metal-free apoenzymes and chromophoric zinc chelators or EDTA, we determined the dissociation constants in the absence and presence of substrates. For the formation of the monozinc enzymes we determined constants of 1.8, 5.1, 0.007, and 2.6 nM in the absence and 13.6, 1.8, 1.2, and 5.7 pM in the presence of substrates for BcII, BlaB, CphA, and L1, respectively. A second zinc ion binds in the absence (presence) of substrates with considerably higher dissociation constants, namely 1.8 (0.8), 0.007 (0.025), 50 (1.9), and 0.006 (0.12) M for BcII, BlaB, CphA, and L1, respectively. We have concluded that the apo form might be the prevailing state of most of the metallo-␤-lactamases under physiological conditions in the absence of substrates. Substrate availability induces a spontaneous self-activation due to a drastic decrease of the dissociation constants, resulting in the formation of active mononuclear enzymes already at picomolar free zinc ion concentrations. In the presence of substrates, the binuclear state of the enzymes only exists at unphysiologic high zinc concentrations and might be of no biological relevance. From the competition experiments with EDTA it is further concluded that the reactivation rate does not depend on the pool of free zinc ions but proceeds via the EDTA-Zn(II)-enzyme ternary complexes.Metallo-␤-lactamases (class B ␤-lactamases) are produced by bacteria as extracellular or periplasmatic enzymes. All known representatives possess two conserved metal binding sites and require zinc ions as enzymatic cofactors. By catalyzing the hydrolysis of ␤-lactams they render the corresponding strains resistant to almost all ␤-lactam antibiotics. Their increasing emergence in pathogenic bacterial strains due to a rapid dissemination by horizontal gene transfer has induced a growing interest in this enzyme family because of the lack of efficient therapies to treat infected patients.The metallo-␤-lactamases constitute a group of heterogeneous proteins that is divided into subclasses B1, B2, and B3 (1). Subclass B1 exhibits a broad substrate profile (2), and its zinc binding sites are composed of His-116, His-118, and His-196 (site 1) and Asp-120, Cys-221, and His-263 (site 2) (numbering according to Ref. 3). In subclass B2 the zinc ligands in site 2 are conserved, whereas His-116 in site 1 is replaced by Asn. Representatives of subclass B2 efficiently hydrolyze only carbapenems (2) and are active as monozinc enzymes, whereas the binding of a second zinc ion causes non-competitive inhibition (4). In subclass B3, Cys-221 is substituted by a Ser and is replaced by His-121 as a zinc ligand in site 2. In the Gob-1 enzyme (Chryseobacterium meningosepticum PINT) an additional H...

Metal Binding Asp-120 in Metallo-β-lactamase L1 from Stenotrophomonas maltophilia Plays a Crucial Role in Catalysis

“…This confirms the proposition of Ullah et al (37), that in L1, both Zn(II)s are necessary for tight substrate binding and in turn full catalytic activity of the enzyme. It should be noted that this result is contrary to recently published work (59) asserting that ␤-lactamases, such as L1 and CcrA (metallo-␤-lactamase from B. fragilis), are apo-(without metal) or mononuclear enzymes in vivo.…”

Glycolytic and Non-glycolytic Functions of Mycobacterium tuberculosis Fructose-1,6-bisphosphate Aldolase, an Essential Enzyme Produced by Replicating and Non-replicating Bacilli

“…Further supporting this reaction mechanism, the same FBP-zinc ion stoichiometry was observed in the structure of the FBA-tb⅐FBP complex (19), suggesting that ligand-activated Zn 2ϩ binding may be a general recruitment mechanism to maximize binding and catalytic activity. Such ligand-activated Zn 2ϩ binding has been reported for metallo-␤-lactamases where the apo form is the prevailing state under physiological conditions in the absence of substrates (69). Substrate availability apparently induces a spontaneous self-activation due to a decrease of the dissociation constants, resulting in the formation of fully active enzyme.…”

Coordination Geometries of Metal Ions in D- or L-Captopril-inhibited Metallo-β-lactamases

“…We have shown earlier that a single metal ion, when bound to the binuclear site, is distributed between both binding sites (10, 12) and that rapid exchange among binding sites occurs (18). A recent work (28) shows that only mononuclear metallo-␤-lactamases might be physiologically important. These findings pose the question whether strategies to find inhibitors for the binuclear enzymes are the only ones being adequate.…”