CHARACTERIZATION OF NATIVE AND RECOMBINANT FORMS OF AN UNUSUAL COBALT-DEPENDENT PROLINE DIPEPTIDASE (PROLIDASE) FROM THE HYPERTHERMOPHILIC ARCHAEON PYROCOCCUS-FURIOSUS

Proline dipeptidase (prolidase) was purified from cell extracts of the proteolytic, hyperthermophilic archaeon Pyrococcus furiosus by multis tep chromatography. The enzyme is a homodimer (39.4 kDa per subunit) a nd as purified contains one cobalt atom per subunit. Its catalytic act ivity also required the addition of Co2+ ions (K-d, 0.24 mM), indicati ng that the enzyme has a second metal ion binding site. Co2+ could be replaced by Mn2+ (resulting in a 25% decrease in activity) but not by Mg2+, Ca2+, Fe2+, Zn2+, Cu2+, or Ni2+. The prolidase exhibited a narro w substrate specificity and hydrolyzed only dipeptides with proline at the C terminus and a nonpolar amino acid (Met, Leu, Val, Phe, or Ala) at the N terminus. Optimal prolidase activity with Met-Pro as the sub strate occurred at a pH of 7.0 and a temperature of 100 degrees C. The N-terminal amino acid sequence of the purified prolidase was used to identify in the P. furiosus genome database a putative prolidase-encod ing gene with a product corresponding to 349 amino acids. This gene wa s expressed in Escherichia coli and the recombinant protein was purifi ed. Its properties, including molecular mass, metal ion dependence, pH and temperature optima, substrate specificity, and thermostability, w ere indistinguishable from those of the native prolidase from P. furio sus. Furthermore, the K-m values for the substrate Met-Pro were compar able for the native and recombinant forms, although the recombinant en zyme exhibited a twofold greater V-max value than the native protein. The amino acid sequence of P. furiosus prolidase has significant simil arity,vith those of prolidases from mesophilic organisms, but the enzy me differs from them in its substrate specificity, thermostability, me tal dependency, and response to inhibitors. The P. furiosus enzyme app ears to be the second Go-containing member (after methionine aminopept idase) of the binuclear N-terminal exopeptidase family.