Highly dynamic metal exchange in anthrax lethal factor involves the occupation of an inhibitory metal binding site

Abstract

Metal exchange is a common strategy to replace the zinc ion of many zinc proteins with other transition metals amenable to spectroscopic investigations. We here demonstrate that in anthrax lethal factor (and likely other zinc proteases), metal exchange is a fast process, and involves the occupation of an inhibitory metal site by the incoming ion prior to the release of zinc.

Identification of an Additional Metal-Binding Site in Human Dipeptidyl Peptidase III

“…The binding site of the catalytic Zn ion in DPP III is very similar to those in thermolysin (TML), carboxypeptidase A (CA), and anthrax lethal factor, i.e., in all of these, the zinc ion is coordinated by two histidines and a glutamate. In addition, all of these proteins can bind other divalent ions, such as Cu, Co, and Mn, excess metal ions inhibit their enzyme activity, and an inhibitory binding site has been identified [ 20 , 22 , 23 ]. It has also been shown that the binding of zinc and other metals in excess can lead to the inhibition of DPP III in humans, rats, and microorganisms [ 9 , 10 , 11 ].…”

“…It has also been shown that the binding of zinc and other metals in excess can lead to the inhibition of DPP III in humans, rats, and microorganisms [ 9 , 10 , 11 ]. All of these findings indicate that metal ions can bind not only at the catalytic site of DPP III, but also at an additional, so-called inhibitory binding site [ 19 , 20 , 21 ].…”

“…There is only one zinc ion in the active site of the crystallographically determined structures of DPP III (PDB IDs of structures of the following: human—3FVY, 3T6B, 3T6J, 5EGY, 5E2Q, 5E33, 5E3A, 5E3C, 5EHH [ 12 , 13 ]; yeast—3CSK [ 14 ], fungal—5YFB, 5YFC, 5YFD [ 15 ]; bacterial DPP III—5NA6, 6NA7, 5NA8, 5ZUM, 6EOM [ 16 , 17 , 18 ]), but previous studies [ 19 , 20 , 21 ] clearly indicated the possibility of another metal-ion binding, which inhibits the enzymatic activity of DPP III. The binding of another metal ion in the so-called inhibitory metal-binding site, which is directly adjacent to the catalytically active site of the enzyme, has been observed in the crystallographic structures of three zinc-dependent enzymes, in which, as in DPP III, the catalytic zinc is coordinated with two histidines and the carboxyl groups of the amino acids Glu or Asp: carboxypeptidase A, thermolysin, and LpxC (the PDB codes of the corresponding structures are 1CPX, 1LND, and 1P42) [ 22 , 23 , 24 ].…”

“…The binding of another metal ion in the so-called inhibitory metal-binding site, which is directly adjacent to the catalytically active site of the enzyme, has been observed in the crystallographic structures of three zinc-dependent enzymes, in which, as in DPP III, the catalytic zinc is coordinated with two histidines and the carboxyl groups of the amino acids Glu or Asp: carboxypeptidase A, thermolysin, and LpxC (the PDB codes of the corresponding structures are 1CPX, 1LND, and 1P42) [ 22 , 23 , 24 ]. Young and Siemann showed that in anthrax lethal factor (LF), metal ions are exchanged in such a way that the binding of the metal to the inhibitory binding site precedes the release of catalytic zinc [ 20 ]. Using the same procedure that Young and Siemann used to determine the potential binding site of the inhibitory Zn 2+ ion in LF, we can determine the binding site of the inhibitory metal in DPP III.…”

Removal of Metal from Carboxypeptidase A Proceeds via a Split Pathway: Implications for the General Mechanisms of Metalloenzyme Inactivation by Chelating Agents

“…Chelators such as 1,10-phenanthroline (OP) and dipicolinic acid (DPA) have been found to follow an A-type mechanism with a variety of metalloenzymes . In contrast, larger and more sterically demanding chelators such as ethylenediaminetetraacetic acid (EDTA) are generally considered to act via a D-type mechanism, ,, and have therefore, been employed to determine (spontaneous) metal dissociation rate constants. , It is important to point out in this connection, however, that some reported observations (such as the dependence of the rate of inactivation on the concentration of EDTA, or the increased protection against inactivation by higher substrate concentrations) have challenged the longstanding assumption that EDTA follows a strictly dissociative mechanism. − …”