Interactions of calcium and other metal ions with caldolysin, the thermostable proteinase from Thermus aquaticus strain T351

Caldolysin, the extracellular proteinase from the extreme thermophile Thermus aquaticus strain T351, is stabilized by Ca2+. A variety of metal ions were able to substitute for Ca2+. Most were unable to confer as much stability as Ca2+, with the exception of the lanthanide ions, which increased the half-life at 95 degrees C from 1 h to more than 4 h. Results from a variety of separation methods indicated that caldolysin binds 6 Ca2+ ions/molecule of enzyme. The presence of non-linear Ca2+ titration plots, and the removal of 4 Ca2+ ions/molecule by treatment with a cationic ion-exchange gel suggested that caldolysin possesses at least two different types of Ca2+-binding sites, with different affinities. Average binding constants of the two types of binding sites were 2.8×10(4)M-1 (for the low-affinity sites) and 7.5×10(5) M-1 (for the high-affinity sites). The total Ca2+-binding free energy for caldolysin was shown to be greater than for either thermolysin or Bacillus subtilis neutral proteinase. It appears that the higher thermostability of caldolysin is due to the presence of 6 Ca2+ ions rather than 4 Ca2+ ions/molecule.

Primary structure of the N-terminal region (1-95) of thermitase, a thermostable proteinase from Thermoactinomyces vulgaris

The 95-residue N-terminal amino acid sequence of thermitase, a thermostable serine proteinase from T. vulgaris, is the following: Tyr-Thr-Pro-Asn-Asp-Pro-Tyr-Phe-Ser-Ser-Arg-Gln-Tyr-Gly-Pro-Gln-Lys-Ile-Gln-Ala-Pro-Gln-Ala-Trp-Asp-Ile-Ala-Glu-Gly-Ser-Gly-Ala-Lys-Ile-Ala-Ile-Val-Asp-Thr-Gly-Val-Gln-Ser-Asn-His-Pro-Asp-Leu-Ala-Gly-Lys-Val-Val-Gly-Gly-Trp-Asp-Phe-Val-Asp-Asn-Asp-Ser-Thr-Pro-Gln-Asn-Gly-Asn-Gly-His-Gly-Thr-His-Cys-Ala-Gly-Ile-Ala-Ala-Ala-Val-Thr-Asn-Asn-Ser-Thr-Gly-Ile-Ala-Gly-Thr-Ala-Pro-Lys. The sequential data were obtained by analyses of peptides isolated from the soluble part of the tryptic digest of the N-terminal cyanogen bromide fragment of the enzyme and from the chymotryptic digest of a thermitase fragment obtained by 2-nitro-5-thiocyanobenzoate cleavage of the enzyme at its single cysteine residue.