CATALYTIC PROPERTIES AND POTENTIAL OF AN EXTRACELLULAR PROTEASE FROM AN EXTREME HALOPHILE

An extracellular protease has been isolated and partially purified fro m the extreme halophile Halobacterium halobium (ATCC 43214). The major enzyme component has a M(r) of 66,000 and is highly dependent upon sa lt concentrations near saturation for catalytic activity and stability . In aqueous solutions, a decrease in the NaCl concentration from 4 to 1 M results in an increase of nearly three orders of magnitude in the first-order rate constant of inactivation at 30 degrees C. Salt effec ts the stability of the enzyme in a cooperative manner, with a Hill co efficient of 4.1, which is similar to that of other enzymes from extre me halophiles. The enzyme activity is dramatically affected by the sal t concentration, with a loss of 2.5 orders of magnitude in k(cat)/K-m in going from 4 to 0 M NaCl. This loss in catalytic efficiency is prim arily due to a dramatic increase in the K-m for the substrate in low-s alt media. Thermodynamic analysis revealed that this K-m increase was mainly the result of increased solubility of the synthetic peptide sub strate in low-salt media, which dramatically increases the ground-stat e stability of the substrate. This results in an effectively reduced s ubstrate partitioning from the bulk solution into the enzyme's active site and an increased value of K-m. The halophilic protease is also ac tive in DMF/water mixtures, albeit with novel catalytic properties. In 33% (v/v) DMF in aqueous buffer, the esterase activity of the enzyme is ca. 80-fold higher than the corresponding amidase activity. This co ntrasts to the situation in pure aqueous buffer, in which the esterase activity is only fourfold higher than the amidase activity. The incre ased esterase activity relative to amidase activity prompted us to inv estigate the use of the protease in kinetically controlled peptide syn thesis. The enzyme has a broad acyl donor substrate specificity and ca n effectively use amino acid esters of Phe, Tyr, Trp, Ser, Gly, and Al a. The enzyme is significantly more selective for the amino acid amide , preferring Gly in the P-1' site. A series of glycine-containing olig opeptides have been prepared in yields up to 76% without degradation d ue to secondary hydrolysis.