Weakly bound calcium ions involved in the thermostability of aqualysin I, a heat-stable subtilisin-type protease of Thermus aquaticus YT-1.
Aqualysin I is a heat-stable protease; in the presence of 1 mM Ca(2+), the enzyme is stable at 80 degrees C and shows the highest activity at the same temperature. After gel filtration to remove free Ca(2+) from the purified enzyme sample, the enzyme (holo-aqualysin I) still bound Ca(2+) (1 mol/mol of the enzyme), but was no longer stable at 80 degrees C. On treatment of the holo-enzyme with EDTA, bound Ca(2+) decreased to about 0.3 mol/mol of the enzyme. The thermostability of holo-aqualysin I was dependent on the concentration of added Ca(2+), and 1 mM added Ca(2+) stabilized the enzyme completely, suggesting that aqualysin I has at least two Ca(2+) binding sites, i.e. stronger and weaker binding ones. Titration calorimetry showed single binding of Ca(2+) to the holo-enzyme with an association constant of 3.1 x 10(3) M(-1), and DeltaH and TDeltaS were calculated to be 2.3 and 6.9 kcal/mol, respectively, at 13 degrees C. La(3+), Sr(2+), Nd(3+), and Tb(3+) stabilized the holo-enzyme at 80 degrees C, as Ca(2+) did. These results suggest that the weaker binding site exhibits structural flexibility to bind several metal cations different in size and valency, and that the metal binding to the weaker binding site is essential for the thermostability of aqualysin I.
Lin
S J
SJ
Department of Biotechnology, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan.
Yoshimura
E
E
Sakai
H
H
Wakagi
T
T
Matsuzawa
H
H
D001665:Binding Sites; D002118:Calcium / Q000737:chemistry* / Q000494:pharmacology; D002122:Calcium Chloride; D002614:Chelating Agents; D002942:Circular Dichroism; D004492:Edetic Acid; D004795:Enzyme Stability / Q000187:drug effects; D006358:Hot Temperature; D008670:Metals; D011489:Protein Denaturation; D012117:Resins, Synthetic; D012697:Serine Endopeptidases / Q000737:chemistry*; D013816:Thermodynamics; D013824:Thermus / Q000201:enzymology*
D002614:Chelating Agents; D008670:Metals; D012117:Resins, Synthetic; C024997:Chelex 100; D004492:Edetic Acid; D012697:Serine Endopeptidases; C055102:aqualysin I; D002122:Calcium Chloride; D002118:Calcium