COLD ADAPTATION OF PROTEINS - PURIFICATION, CHARACTERIZATION, AND SEQUENCE OF THE HEAT-LABILE SUBTILISIN FROM THE ANTARCTIC PSYCHROPHILE BACILLUS TA41

The gene of subtilisin S41, an alkaline protease secreted by the psych rophile Bacillus TA41, encodes for a preproenzyme of 419 amino acids r esidues. The nucleotide sequence and NH2- and COOH-terminal amino acid sequencing of the purified enzyme indicate that the mature subtilisin S41 is composed of 309 residues with a predicted M(r) = 31,224. Subti lisin S41 shares most of its properties with mesophilic subtilisins (s tructure of the precursor, 52% amino acid sequence identity, alkaline pH optimum, broad specificity, Ca2+ binding) but is characterized by a higher specific activity on macromolecular substrate, by a shift of t he optimum of activity toward low temperatures, and by a low thermal s tability. The enzyme also differs by an acidic pI (5.3) and the presen ce of one disulfide bond. It is proposed that the psychrophilic enzyme possesses a more flexible molecular structure when compared to mesoph ilic and thermophilic subtilases in order to compensate for the reduct ion of reaction rates at low temperatures. The model of subtilisin S41 indeed reveals several features able to induce a more flexible, heat- labile conformation: the occurrence of four extended surface loops, a very hydrophilic surface through 11 extra Asp residues, and the lack o f several salt bridges and aromatic-aromatic interactions. The low aff inity of the Ca1 calcium binding site (K-d(app) = 10(-6) M), resulting possibly from one chelating side chain substitution and the stacking of Gly residues, also reflect a less compact conformation. The differe nce of free energy of stabilization between subtilisin S41 and a mesop hilic subtilisin suggests that the balance of exo and endothermically formed weak bonds is critical for the enzyme flexibility.