A novel metallocarboxypeptidase (PfuCP) has been
purified to homogeneity from the hyperthermophilic archaeon,
Pyrococcus furiosus, with its intended use in
C-terminal ladder sequencing of proteins and peptides at
elevated temperatures. PfuCP was purified in its inactive
state by the addition of ethylenediaminetetraacetic acid
(EDTA) and dithiothreitol (DTT) to purification buffers,
and the activity was restored by the addition of divalent
cobalt (Kd = 24 ± 4 μM at
80 °C). The serine protease inhibitor phenylmethylsulfonyl
fluoride (PMSF) had no effect on the activity. The molecular
mass of monomeric PfuCP is 59 kDa as determined by matrix-assisted
laser desorption ionization time-of-flight mass spectrometry
(MALDI-TOF MS) and 58 kDa by SDS-PAGE analysis. In solution,
PfuCP exists as a homodimer of ∼128 kDa as determined
by gel filtration chromatography. The activity of PfuCP
exhibits a temperature optimum exceeding 90 °C under
ambient pressure, and a narrow pH optimum of 6.2–6.6.
Addition of Co2+ to the apoPfuCP at
room temperature does not alter its far-UV circular dichroism
(CD) or its intrinsic fluorescence spectrum. Even when
the CoPfuCP is heated to 80 °C, its far-UV CD shows
a minimal change in the global conformation and the intrinsic
fluorescence of aromatic residues shows only a partial
quenching. Changes in the intrinsic fluorescence appear
essentially reversible with temperature. Finally, the far-UV
CD and intrinsic fluorescence data suggest that the overall
structure of the holoenzyme is extremely thermostable.
However, the activities of both the apo and holo
enzyme exhibit a similar second-order decay over time,
with 50% activity remaining after ∼40 min at 80 °C.
The N-blocked synthetic dipeptide, N-carbobenzoxy-Ala-Arg
(ZAR), was used in the purification assay. The kinetic
parameters at 80 °C with 0.4 mM CoCl2 were:
Km, 0.9 ± 0.1 mM;
Vmax, 2,300 ± 70 U
mg−1; and turn over number, 600 ±
20 s−1. Activity against 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 residues. This broad specificity was confirmed by
the C-terminal ladder sequencing of several synthetic and
natural peptides, including porcine N-acetyl-renin substrate,
for which we have observed (by MALDI-TOF MS) stepwise hydrolysis
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.