Able to reach and remove the active site metal by

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able to reach and remove the active site metal by slowly diffusing in the crystals through the hydration water, while the apoprotein is maintained in the native conformation by the crystal packing forces and denaturation is avoided. After the chelating agent is dialyzed out, often against a high-salt (e.g., CIOi) buffer to reduce nonspecific binding, a new metalloprotein can obtained by addition of the appropriate metal salt. 23 Cobalt(II)-substituted zinc proteins often show about as much activity as the native zinc enzymes (Table 2.4). This is a general characteristic of the cobalt- substituted zinc enzymes,24 since the coordination chemistry of cobalt(II) is very similar to that of zinc(II). The two ions also show virtually identical ionic radii. Cobalt(II) derivatives generally display useful electronic spectra. High-spin co- balt(II) ions are paramagnetic, containing three unpaired electrons (5 = ~); thus they can also give rise to EPR spectra. The electronic relaxation times, i.e., the average lifetimes of the unpaired electrons in a given spin state of the 5 mani- fold ( -!, - i, i, ~), are very short (lO - 11 to 10 - J 2 s) at room temperature. In order to detect EPR spectra, the sample temperature is usually decreased, often down to liquid helium temperature, to increase the electronic relaxation times and sharpen the EPR linewidths. On the other hand, as the paramagnetic broad- ening of the NMR lines in such systems is inversely proportional to the elec- tronic relaxation times (see Section IV.C.3), room-temperature IH NMR spectra of cobalt(II) complexes can be easily detected, even in the absence of chemical exchange. Therefore cobalt(II) is an exceptional probe to monitor the structure
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46 2 I THE REACTION PATHWAYS OF ZINC ENZYMES AND RELATED BIOLOGICAL CATALYSTS and reactivity of zinc enzymes. Of course, the transfer of information from the artificial to the native enzyme must be done with cauti{)n. However, if we can understand the functioning of the cobalt enzyme, we then have a reference frame by which to understand the kinetic properties of the native enzyme. The spec- troscopic properties of cobalt(Il) in cobalt-substituted proteins have been re- viewed. 25 Copper(Il)-substituted zinc proteins are generally inactive with respect to the natural and most artificial substrates (Table 2.4). In model compounds cop- per(Il) is often principally four-coordinate, with at most two more ligands pre- sent at metal-ligand distances that are longer than normal coordination bonds. As a consequence, the ability of zinc to switch between four- and five-coordi- nate species without any appreciable barrier and with usual metal-donor dis- tances is not mimicked by copper. Furthermore, binding at the four principal coordination positions is generally stronger for copper than for zinc. It follows that substrates may have slow detachment kinetics. These properties are unfa- vorable for catalysis.
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