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Originally published In Press as doi:10.1074/jbc.M102659200 on August 28, 2001

J. Biol. Chem., Vol. 276, Issue 43, 39629-39637, October 26, 2001
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Kinetic Analysis of the Conjugation of Ubiquitin to Picornavirus 3C Proteases Catalyzed by the Mammalian Ubiquitin-protein Ligase E3alpha *

T. Glen LawsonDagger §, Molly E. SweepDagger , Peter E. SchlaxDagger , Richard N. Bohnsack, and Arthur L. Haas

From the Dagger  Department of Chemistry, Bates College, Lewiston, Maine 04240 and the  Department of Biochemistry, Medical College of Wisconsin, Milwaukee, Wisconsin 53226

The 3C proteases of the encephalomyocarditis virus and the hepatitis A virus are both type III substrates for the mammalian ubiquitin-protein ligase E3alpha . The conjugation of ubiquitin to these proteins requires internal ten-amino acid-long protein destruction signal sequences. To evaluate how these destruction signals modulate interactions that must occur between E3alpha and the 3C proteases, we have kinetically analyzed the formation of ubiquitin-3C protease conjugates in a reconstituted system of purified E1, HsUbc2b/E214Kb, and human E3alpha . Our measurements show that the encephalomyocarditis virus 3C protease is ubiquitinated in this system with Km = 42 ± 11 µM and Vmax = 0.051 ± 0.01 pmol/min whereas the parameters for the ubiquitination of the hepatitis A virus 3C protease are Km = 20 ± 5 µM and Vmax = 0.018 ± 0.003 pmol/min. Mutations in the destruction signal sequences resulted in changes in the rate at which E3alpha conjugates ubiquitin to the altered 3C protease proteins. The Km and Vmax values for these reactions change proportionally in the same direction. These results suggest differences in rates of conjugation of ubiquitin to 3C proteases are primarily a kcat effect. Replacing specific encephalomyocarditis virus 3C protease lysine residues with arginine residues was found to increase, rather than decrease, the rate of ubiquitin conjugation, and the Km and Vmax values for these reactions are both higher than for the wild type protein. The ability of E3alpha to catalyze the conjugation of ubiquitin to both 3C proteases was found to be inhibited by lysylalanine and phenylalanylalanine, demonstrating that the same sites on E3alpha that bind destabilizing N-terminal amino acids in type I and II substrates also interact with the 3C proteases.


* This work was supported in part by National Science Foundation Research in Undergraduate Institutions Award MCB-9974497 (to T. G. L.), a Henry Dreyfus Teacher Scholar Award (to T. G. L.), a Roger C. Schmutz Faculty Research Grant (to T. G. L.), and by United States Public Health Services Grant GM 34009 (to A. L. H.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ To whom correspondence should be addressed. Tel.: 207-786-6293; Fax: 207-786-8336; E-mail: tlawson@bates.edu.


Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.


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T. J. Siepmann, R. N. Bohnsack, Z. Tokgoz, O. V. Baboshina, and A. L. Haas
Protein Interactions within the N-end Rule Ubiquitin Ligation Pathway
J. Biol. Chem., March 7, 2003; 278(11): 9448 - 9457.
[Abstract] [Full Text] [PDF]




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