Cheng, Timothy C. and Ramakrishnan, Vijay and Chan, Sunney I. (1999) Purification and characterization of a cobalt-activated carboxypeptidase from the hyperthermophilic archaeon Pyrococcus furiosus. Protein Science, 8 (11). pp. 2474-2486. ISSN 0961-8368. http://resolver.caltech.edu/CaltechAUTHORS:20120110-102835248
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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 (K_d = 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 Co^(2+) 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 CoCl_2 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 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-z Phe-His-Leu-Leu-Val-Tyr-Ser.
|Additional Information:||© 1999 The Protein Society. Article first published online: 31 Dec 2008. Received May 7, 1999; Accepted August 13, 1999. This research was supported in part by NIH grant GM 22432 from the National Institute of General Medical Sciences, US Public Health Service and grant NSC 88-2113-M-001-037 from the National Science Council, Taiwan. We gratefully acknowledge the technical support of Phil Johnson at the University of Wisconsin Fermentation Facility (Madison, Wisconsin) for assistance in the culturing of P. furiosus cells. N-terminal Edman sequencing and MALDI-TOF mass analysis were carried out at the Caltech Protein and Peptide Microanalytical Laboratory under the direction of Gary M. Hathaway. The MALDI-TOF mass spectrometer was purchased with funds awarded by NIH Shared Instrumentation Grant RR11292.|
|Subject Keywords:||C-terminal sequencing; carboxypeptidase; extremozyme; hyperthermophile; Pyrococcus furiosus|
|Official Citation:||Cheng, T. C., Ramakrishnan, V. and Chan, S. I. (1999), Purification and characterization of a cobalt-activated carboxypeptidase from the hyperthermophilic archaeon Pyrococcus furiosus. Protein Science, 8: 2474–2486. doi: 10.1110/ps.8.11.2474|
|Usage Policy:||No commercial reproduction, distribution, display or performance rights in this work are provided.|
|Deposited By:||Ruth Sustaita|
|Deposited On:||10 Jan 2012 19:13|
|Last Modified:||10 Jan 2012 19:13|
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