Kinetic analysis of the conjugation of ubiquitin to picornavirus 3C proteases catalyzed by the mammalian ubiquitin-protein ligase E3 alpha

The 3C proteases of the encephalomyocarditis virus and the hepatitis A viru s are both type III substrates for the mammalian ubiquitin-protein ligase E 3 alpha. The conjugation of ubiquitin to these proteins requires internal t en-amino acid-long protein destruction signal sequences. To evaluate how th ese destruction signals modulate interactions that must occur between E3a a nd the 3C proteases, we have kinetically analyzed the formation of ubiquiti n-3C protease conjugates in a reconstituted system of purified E1, HsUbc2b/ E2(14Kb), and human E3a. Our measurements show that the encephalomyocarditi s virus 3C protease is ubiquitinated in this system with K-m = 42 +/- 11 mu M and V-max = 0.051 +/- 0.01 pmol/min whereas the parameters for the ubiqui tination of the hepatitis A virus 3C protease are K-m = 20 +/- 5 muM and V- max = 0.018 +/- 0.003 pmol/min. Mutations in the destruction signal sequenc es resulted in changes in the rate at which E3a conjugates ubiquitin to the altered 3C protease proteins. The K-m and V-max values for these reactions change proportionally in the same direction. These results suggest differe nces in rates of conjugation of ubiquitin to 3C proteases are primarily a k (cat) effect. Replacing specific encephalomyocarditis virus 3C protease lys ine residues with arginine residues was found to increase, rather than decr ease, the rate of ubiquitin conjugation, and the K,, and V.. values for the se reactions are both higher than for the wild type protein. The ability of E3a to catalyze the conjugation of ubiquitin to both 3C proteases was foun d to be inhibited by lysylalanine and phenylalanylalanine, demonstrating th at the same sites on E3a that bind destabilizing N-terminal amino acids in type I and 11 substrates also interact with the 3C proteases.