Biochemical and kinetic characterization of metallopeptidases from the hyperthermophilic archaeon Pyrococcus furiosus
Story, Sherry Venette
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The primary goal of this research project was to purify and characterize the biochemical and kinetic properties of three distinct zinc metallopeptidases and try to determine the physiological role of these enzymes in Pyrococcus furiosus. These enzymes include an aminoacylase, a lysine aminopeptidase, and an alanine aminopeptidase. None of these enzymes had been isolated or characterized from a hyperthermophile or from an archaeon, at the onset of this project. Aminoacylase catalyzes the hydrolysis of nonpolar N-acylated-L-amino acids (Met, Ala, Val, and Leu) and was purified by multistep chromatography. It contains three zinc atoms per subunit and is optimally active at 100°C. The gene (PF0597) encoding the enzyme was cloned but active recombinant enzyme was not produced in Escherichia coli. Based on its substrate specificity and kinetic properties, aminoacylase is proposed to have a role in peptide catabolism of P. furiosus Lysine aminopeptidase (KAP) was purified by multistep chromatography from cell extracts of P. furiosus. It contains two zinc atoms per subunit and hydrolyzed only basic N-terminal residues (Lys- and Arg-pNA). Surprisingly, its activity was stimulated four-fold by the addition of cobalt ions. The gene (PF1861) encoding the enzyme was was expressed in Escherichia coli and the recombinant protein was purified. Its properties, including molecular mass, metal ion dependence, pH and temperature optima were indistinguishable from those of the native form. Based on its amino acid sequence, KAP is annotated in the genome sequence as an endoglucancase, although such an activity could not be demonstrated, KAP is part of the M18 family of peptidases which includes yeast aminopeptidase I. KAP is the first lysine aminopeptidase to be purified from a prokaryotic source. Another gene (PF0369) annotated in the genome of Pyrococcus furiosus as encoding an endoglucanase was cloned and expressed in Escherichia coli. The recombinant enzyme also lacked endoglucanase activity and hydrolyzed tri- and tetrapeptides containing an Ala residue at the N-terminus indicating that it is an alanine aminopeptidase. It has an optimal temperature for activity of 100 oC using Ala-Ala-Ala-Ala [2mM] as the substrate. The enzyme contains two zinc atoms per subunit but its activity was strictly dependent on the addition of cobalt ions for reasons not known. The kinetic properties of both alanine and leucine aminopeptidase suggest that these two enzymes play roles in the degradation of peptide growth substrates of P. furiosus.