Thermolysin calcium ions

In thermolysin (Matthews et al., 1974 Holmes and Matthews, 1982) zinc is bound approximately tetrahedrally to glutamate (monodentate), two histidines, and water. While zinc in the native enzyme is tetracoord-inate, in some inhibitor complexes it is pentacoordinate. The four calcium ions are bound by six to eight oxygen ligands, as shown in Fig. 35, with Ca 0 distances of 2.23-2.71 A. The RNA polymerase from E. coli contains Zn(II) and Mg(II), which may be substituted by Mn(II) (Chuk-nyhVy et al., 1990). 

Figure 10-3. The environment of the four structural calcium ions in the enzyme thermolysin isolated from Bacillus thermoproteolyticus. All of the calcium ions are associated with oxygen donor ligands.

It should be mentioned here that in certain areas of the life sciences, the use of the word ligand has also been introduced for a (small) substrate that binds to a larger molecule, like DNA or a protein. In such cases, Ca, for example, can be a ligand for the protein thermolysin to stabilize its structure however, at the same time the carboxylate side chains of the thermolysin backbone are ligands for the calcium ions In this chapter, the use of ligand will only be in combination with a metal. 

Thermolysin (EC 3.4.24.4) a heat-stable, zinc- and calcium-containing neutral protease, M, 37,500, from Bacillus thermoproteolyticus, with a substrate specificity similar to that of Subtilisin (see). After one hour at 80 °C, T. still has 50% original activity. This high heat stability of T. is attributed to the large number of hydrophobic regions and the presence of four bound calcium ions, which serve in place of disulfide bridges (T. contains no disulfide bridges) to maintain the compact shape of the molecule. T. is neither a thiol nor a serine enzyme. 

Matthews et al. have determined the structure of the extracellular proteolytic enzyme, thermolysin, to 2.3 A resolution. This enzyme of molecular weight 37 500 contains one zinc and four calcium ions, and is interesting because of its imusual heat stability. The active site contains a zinc atom tetrahedrally co-ordinated to His-142, His-146, Glu-166, and a water molecule, and in this way resembles carboxypeptidase A. The precise details of the co-ordination of the calcium ions are not reported. However, it is of interest that two have a centre-to-centre distance of 3.8 A and are located within an interior region of the protein, surrounded by carbonyl and carboxylate groups. Loss of calcium does not hinder proteolysis at room temperature, but the enzyme is no longer heat stable. Preliminary X-ray data are also reported for the acidic protease from the fungus Rhizopus chinensis. ... 

Substitution of Ca2+ by Ln3+ is achieved by soaking calcium containing crystals of the macromolecule in a solution of a lanthanide ion. Soaking times are two days in the case of thermolysin [79] and four weeks in the case lysozome [80]. 

In metalloproteins, the metal cofactor may be removed and replaced by a heavier atom with similar chemistry. The zinc in insulin was successfully replaced by cadmium or lead, the zinc in carboxypeptidase and carbonic anhydrase by mercury, the zinc in thermolysin by lanthanide ions or strontium or barium and the calcium in staphylococcal nuclease by barium. Success has most usually been achieved by soaking the crystals in a chelating agent and subsequently diffusing in the heavier atom. 

Horrocks WD Jr, Rhee M-J, Snyder AP, Sudnick DR. leaser-induced metal ion luminescence interlanthanide ion energy transfer distance measurements in the calcium-binding proteins, parvalbiunin, and thermolysin. Metalloprotein models address a photophysical problem. J Am Chem Soc. 1980 102(10) 3650 3652. 

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