One high affinity (nM) and one low affinity (M) macroscopic dissociation constant for the binding of metal ions were found for the wild-type metallo--lactamase from Bacillus cereus as well as six single-site mutants in which all ligands in the two metal binding sites were altered. Surprisingly, the mutations did not cause a specific alteration of the affinity of metal ions for the sole modified binding site as determined by extended x-ray absorption fine structure (EXAFS) and perturbed angular correlation of ␥-rays spectroscopy, respectively. Also UV-visible absorption spectra for the mono-cobalt enzymes clearly contain contributions from both metal sites. The observations of the very similar microscopic dissociation constants of both binding sites in contrast to the significantly differing macroscopic dissociation constants inevitably led to the conclusion that binding to the two metal sites exhibits negative cooperativity. The slow association rates for forming the binuclear enzyme determined by stopped-flow fluorescence measurements suggested that fast metal exchange between the two sites for the mononuclear enzyme hinders the binding of a second metal ion. EXAFS spectroscopy of the mono-and di-zinc wild type enzymes and two di-zinc mutants provide a definition of the metal ion environments, which is compared with the available x-ray crystallographic data.Two zinc binding sites in close proximity are conserved in all metallo--lactamases studied so far. Only two of the metal ion ligands undergo variations between the three different subclasses of the enzyme family (1). The enzyme from Bacillus cereus 569/H/9 (BcII) 1 represents a member of subclass B1 with 3 His ligands in one site and 1 Asp, 1 Cys, and 1 His ligand in the other site (3H 1 and DCH 1 sites, respectively). Various crystal structures of BcII are available, representing mononuclear (2) and binuclear species (3, 4). It was shown earlier that both mono-and binuclear zinc enzymes from B. cereus (5) and Bacteroides fragilis (6) are catalytically active.Although catalytic mechanisms for the enzyme with either one or two zinc ions bound have been discussed (for review see Ref. 7) the respective roles of the two binding sites during catalysis are still unclear. Generally the 3H site is considered to be the primary catalytic site. However, the importance of the DCH site for catalysis became obvious from studies of the C168A mutant. When only one zinc ion is bound to this mutant, it shows a very low activity compared with the wild type, whereas wild type-like activity is almost restored when a second metal ion is bound (5).Perturbed angular correlation (PAC) of ␥-ray spectroscopy provides information on the metal ion coordination geometry through measurement of the nuclear quadrupole interaction (NQI) between the nuclear electric quadrupole moment and the electric field gradient from the surrounding charge distribution. With this method it was possible to demonstrate that the Cd(II) ions in the mononuclear wild type BcII are distributed between the two m...
Extended x-ray absorption fine structure (EXAFS) spectroscopy was combined with thermodynamic and kinetic approaches to investigate zinc binding to a zinc finger (C 2 H 2 ) and a tetrathiolate (C 4 ) peptide. Both peptides represent structural zinc sites of proteins and rapidly bind a single zinc ion with picomolar dissociation constants. In competition with EDTA the transfer of peptide-bound zinc ions proved to be 6 orders of magnitude faster than predicted for a dissociation-association mechanism thus requiring ligand exchange mechanisms via peptide-zinc-EDTA complexes. EXAFS spectra of C 2 H 2 showed the expected Cys 2 His 2 -ligand geometry when fully loaded with zinc. For a 2-fold excess of peptide, however, the existence of zinc-bridged peptidepeptide complexes with dominating sulfur coordination could be clearly shown. Whereas zinc binding kinetics of C 2 H 2 appeared as a simple second order process, the suggested mechanism for C 4 comprises a zinc-bridged Zn-(C 4 ) 2 species as well as a Zn-C 4 species with less than 4 metal-bound thiolates, which is supported by EXAFS results. A rapid equilibrium of bound and unbound states of individual ligands might explain the kinetic instability of zinc-peptide complexes, which enables fast ligand exchange during the encounter of occupied and unoccupied acceptor sites. Depending on relative concentrations and stabilities, this results in a rapid transfer of zinc ions in the virtual absence of free zinc ions, as seen for the zinc transfer to EDTA, or in the formation of zinc-bridged complexes, as seen for both peptides with excess of peptides over available zinc.Within the last decade the generally accepted roles of protein-bound zinc in catalysis and structure stabilization were complemented by regulatory functions. Protein binding sites have been classified as "catalytic," "co-catalytic," and "structural" zinc sites (1). A fourth type, namely "interface" was added recently (2). The possible role of a variable zinc occupancy of protein sites as a modification that provides a pathway for intracellular information transfer has been discussed (3), and the steadily growing number of known zinc proteins involved in gene regulation led to the suggestion that some classes of proteins might transduce changes in available zinc levels into changes in patterns of gene expression (4). An update of recent findings in zinc biology (5) now supports the view of zinc as a key element in cellular regulation.An important question with respect to a regulatory function, however, concerns the controversially discussed concentration of free zinc ions in different physiological environments. Cells react on a changed zinc supply by e.g. an activated transcription of metallothionein (Ref. 6 and references therein) or changes in the activity of zinc-sensing transcription activators like Zap1 (7) and MTF-1 (8). In the latter cases zinc binding to individual zinc finger motifs is supposed to induce structural changes, which in turn modify the functionality of the proteins. If the transduction ...
Two metal ion binding sites are conserved in metallo-beta-lactamase from Aeromonas hydrophila. The ligands of a first zinc ion bound with picomolar dissociation constant were identified by EXAFS spectroscopy as one Cys, two His and one additional N/O donor. Sulfur-to-metal charge transfer bands are observed for all mono- and di-metal species substituted with Cu(II) or Co(II) due to ligation of the single conserved cysteine residue. Binding of a second metal ion results in non-competitive inhibition which might be explained by an alternative kinetic mechanism. A possible partition of metal ions between the two binding sites is discussed.
The present study demonstrates that both the nature (Zn(II), Cd(II) or Hg(II)) and supply of metal ions determine whether zinc fingers fold into the well-known, fully loaded structures or alternatively populate a variety of structural states under substoichiometric conditions. Metal-bridged species are observed by perturbed angular correlation (PAC), EXAFS, UV spectroscopy, and stopped-flow kinetics. Transitions between structural states as adaptive reactions to changed metal-ion supply might represent intelligent system changes in zinc homeostasis, trafficking and signalling, and reflect features of heavy-metal toxicity at the molecular level. Because the zinc fingers exist in structural states that are different from the metal-free and fully loaded species, the prevailing view on metal-mediated molecular regulation in terms of "on and off control" might be oversimplified.
Transient kinetics of the equine infectious anemia virus deoxyuridine 5P P-triphosphate nucleotide hydrolase were characterized by monitoring the fluorescence of the protein. Rate constants for the association and dissociation of substrate and inhibitors were determined and found to be consistent with a onestep mechanism for substrate binding. A C-terminal part of the enzyme presumed to be flexible was removed by limited trypsinolysis. As a result, the activity of the dUTPase was completely quenched, but the rate constants and fluorescent signal of the truncated enzyme were affected only to a minor degree. We conclude that the flexible C-terminus is not a prerequisite for substrate binding, but indispensable for catalysis.z 2000 Federation of European Biochemical Societies.
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