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 Ca2+, the en zyme is stable at 80 degrees C and shows the highest activity at the same t emperature. After gel filtration to remove free Ca2+ from the purified enzy me sample, the enzyme (holo-aqualysin I) still bound Ca2+ (1 mol/mol of the enzyme), but was no longer stable at 80 degrees C. On treatment of the hol o-enzyme with EDTA, bound Ca2+ decreased to about 0.3 mol/mol of the enzyme . The thermostability of holo-aqualysin I was dependent on the concentratio n of added Ca2+, and 1 mM added Ca2+ stabilized the enzyme completely, sugg esting that aqualysin I has at least two Ca2+ binding sites, i.e. stronger and weaker binding ones. Titration calorimetry showed single binding of Ca2 + to the holo-enzyme with an association constant of 3.1 x 10(3) M-1, and D elta H and T Delta S were calculated to be 2.3 and 6.9 kcal/mol, respective ly, at 13 degrees C. La3+, Sr2+, Nd3+, and Tb3+ stabilized the hole-enzyme at 80 degrees C, as Ca2+ did. These results suggest that the weaker binding site exhibits structural flexibility to bind several metal cations differe nt in size and valency, and that the metal binding to the weaker binding si te is essential for the thermostability of aqualysin I. (C) 1999 Elsevier S cience B.V. All rights reserved.