Purification and characterization of a cobalt-activated carboxypeptidase from the hyperthermophilic archaeon Pyrococcus furiosus

A novel metallocarboxypeptidase (PfuCP) has been purified to homogeneity fr om the hyperthermophilic archaeon, Pyrococcus furiosus, with its intended u se in C-terminal ladder sequencing of proteins and peptides at elevated tem peratures. PfuCP was purified in its inactive state by the addition of ethy lenediaminetetraacetic acid (EDTA) and dithiothreitol (DTT) to purification buffers, and the activity was restored by the addition of divalent cobalt (K-d = 24 +/- 4 mu M at 80 degrees C). The serine protease inhibitor phenyl methylsulfonyl fluoride (PMSF) had no effect on the activity. The molecular mass of monomeric PfuCP is 59 kDa as determined by matrix-assisted laser d esorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) and 58 kDa by SDS-PAGE analysis. In solution, PfuCP exists as a homodimer of simi lar to 128 kDa as determined by gel filtration chromatography. The activity of PfuCP exhibits a temperature optimum exceeding 90 degrees C under ambie nt pressure, and a narrow pH optimum of 6.2-6.6. Addition of Co2+ to the ap oPfuCP at room temperature does not alter its far-UV circular dichroism (CD ) or its intrinsic fluorescence spectrum. Even when the CoPfuCP is heated t o 80 degrees C, its far-UV CD shows a minimal change in the global conforma tion and the intrinsic fluorescence of aromatic residues shows only a parti al quenching. Changes in the intrinsic fluorescence appear essentially reve rsible with temperature. Finally, the far-UV CD and intrinsic fluorescence data suggest that the overall structure of the holoenzyme is extremely ther mostable. However, the activities of both the apo and hole enzyme exhibit a similar second-order decay over time, with 50% activity remaining after si milar to 40 min at 80 degrees C. The N-blocked synthetic dipeptide, N-carbo benzoxy-Ala-Arg (ZAR), was used in the purification assay. The kinetic para meters at 80 degrees C with 0.4 mM CoCl2 were. K-m, 0.9 +/- 0.1 mM; V-max, 2,300 +/- 70 U mg(-1); and turn over number, 600 +/- 20 s(-1). Activity aga inst other ZAX substrates (X = V, L, I, M, W, Y, F, N. A, S, H, K) revealed a broad specificity for neutral, aromatic, polar, and basic C-terminal res idues. This broad specificity was confirmed by the C-terminal ladder sequen cing of several synthetic and natural peptides, including porcine N-acetyl- renin substrate, for which we have observed (by MALDI-TOF MS) stepwise hydr olysis by PfuCP of up to seven residues from the C-terminus: Ac-Asp-Arg-Val -Tyr-Ile-His-Pro-Phe-His-Leu-Leu-Val-Tyr-Ser.