Substrate-activated zinc binding of metallo-beta -lactamases: physiological importance of mononuclear enzymes.
We have investigated the influence of substrate binding on the zinc ion affinity of representatives from the three metallo-beta-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) microm for BcII, BlaB, CphA, and L1, respectively. We have concluded that the apo form might be the prevailing state of most of the metallo-beta-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.
Wommer
Sandra
S
Department of Biochemistry, University of Saarland, D-66041 Saarbrücken, Germany.
Rival
Sandrine
S
Heinz
Uwe
U
Galleni
Moreno
M
Frere
Jean-Marie
JM
Franceschini
Nicola
N
Amicosante
Gianfranco
G
Rasmussen
Bodil
B
Bauer
Rogert
R
Adolph
Hans-Werner
HW
D000333:Aeromonas / Q000201:enzymology; D001409:Bacillus cereus / Q000201:enzymology; D002614:Chelating Agents / Q000494:pharmacology; D004492:Edetic Acid / Q000494:pharmacology; D005417:Flavobacterium / Q000201:enzymology; D007700:Kinetics; D011485:Protein Binding; D014974:Xanthomonas / Q000201:enzymology; D015032:Zinc / Q000494:pharmacology*; D001618:beta-Lactamases / Q000737:chemistry* / Q000378:metabolism*
D002614:Chelating Agents; D004492:Edetic Acid; D001618:beta-Lactamases; D015032:Zinc