PURIFICATION AND CHARACTERIZATION OF 2 FUNCTIONAL FORMS OF INTRACELLULAR PROTEASE PFPI FROM THE HYPERTHERMOPHILIC ARCHAEON PYROCOCCUS-FURIOSUS

The hyperthermophilic archaeon Pyrococcus furiosus grows optimally at 100 degrees C by the fermentation of peptides and carbohydrates. From this organism, we have purified to homogeneity an intracellular protea se, previously designated PfpI (P. furiosus protease I) (S. B. Halio, I. I. Blumentals, S. A, Short, B. M. Merrill, and R. M. Kelly, J. Bact eriol. 178:2605-2612, 1996), The protease contains a single subunit wi th a molecular mass of approximately 19 kDa and exists in at least two functional conformations, which were purified separately, The predomi nant form from the purification (designated PfpI-C1) is a hexamer with a molecular mass of 124 +/- 6 kDa (by gel filtration) and comprises a bout 90% of the total activity. The minor form (designated PfpI-C2) is trimeric with a molecular mass of 59 +/- 3 kDa. PfpI-C1 hydrolyzed bo th basic and hydrophobic residues in the P1 position, indicating tryps in- and chymotrypsin-like specificities, respectively. The temperature optimum for Ala-Ala-Phe-7-amido-4-methylcoumarin (AAF-MCA) hydrolysis was similar to 85 degrees C both for purified PfpI-C1 and for proteol ytic activity in P. fitriosus cell extract, In contrast, the temperatu re optimum for PfpI prepared by incubating a cell extract of P. furios us at 98 degrees C in 1% sodium dodecyl sulfate for 24 h at 95 to 100 degrees C (I. I, Blumentals, A. S. Robinson, and R. M. Kelly, Appl, En viron. Microbiol. 56:1255-1262, 1990), designated PfpI-H, was similar to 100 degrees C, Moreover, the half-life of activity of PfpI-C1 at 98 degrees C was less than 30 min, in contrast to a value of more than 3 3 h measured for PfpI-H. PfpI-C1 appears to be a predominant serine-ty pe protease in cell extracts but is converted in vitro, probably in pa rt by deamidation of Asn and Gin residues, to a more thermally stable form (PfpI-H) by prolonged heat treatment, The deamination hypothesis is supported by the differences in the measured pI values of PfpI-C1 ( 6.1) and PfpI-H (3.8). High levels of potassium phosphate (>0.5 mM) we re found to extend the half-life of PfpI-C1 activity towards AAF-MCA b y up to 2.5-fold at 90 degrees C, suggesting that compatible solutes p lay an important role in the in vivo function of this protease.