Purification and characterization of Ak.1 protease, a thermostable subtilisin with a disulphide bond in the substrate-binding cleft.
Ak.1 protease, a thermostable subtilisin isolated originally from Bacillus st. Ak.1, was purified to homogeneity from the Escherichia coli clone PB5517. It is active against substrates containing neutral or hydrophobic branched-chain amino acids at the P(1) site, such as valine, alanine or phenylalanine. The K(m) and k(cat) of the enzyme decrease with decreasing temperature, though not to the same degree with all substrates, suggesting that specificity changes with temperature. The protease is markedly stabilized by Ca(2+) ions. At 70 degrees C, a 10-fold increase in Ca(2+) concentration increases the half-life by three orders of magnitude. Ak.1 protease is stabilized by Ca(2+) to a greater extent than is thermitase. This may be due, in part, to the presence of an extra Ca(2+)-binding site in Ak.1 protease. Other metal ions, such as Sr(2+), increase the thermostability of the enzyme, but to a significantly lower degree than does Ca(2+). The structure of the protease showed the presence of a disulphide bond located within the active-site cleft. This bond influences both enzyme activity and thermostability. The disulphide bond appears to have a dual role: maintaining the integrity of the substrate-binding cleft and increasing the thermostability of the protease. The protease was originally investigated to determine its usefulness in the clean-up of DNA at high temperatures. However, it was found that this protease has a limited substrate specificity, so this application was not explored further.
Thermophile Research Unit, University of Waikato, Private Bag 3105, Hamilton 2001, New Zealand. thermlit@exeter.ac.uk
H S
HS
Toogood
C A
CA
Smith
E N
EN
Baker
R M
RM
Daniel
D004220:Disulfides / Q000378:metabolism*; D004795:Enzyme Stability; D006358:Hot Temperature; D011487:Protein Conformation; D013379:Substrate Specificity; D013381:Subtilisins / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism
D004220:Disulfides; D013381:Subtilisins