The 3C proteases of the encephalomyocarditis virus and the hepatitis A virus are both type III substrates for the mammalian ubiquitin-protein ligase E3. 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 E3 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 E3. Our measurements show that the encephalomyocarditis virus 3C protease is ubiquitinated in this system with Km = 42 + or - 11 µM and Vmax = 0.051 + or - 0.01 pmol/min while the parameters for the ubiquitination of the hepatitis A virus 3C protease are Km = 20 + or - 5 µM and Vmax = 0.018 + or - 0.003 pmol/min. Mutations in the destruction signal sequences resulted in changes in the rate at which E3 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 EMCV 3C protease lysine residues with arginine residues was found to increase, rather than decrease, the rate of ubiquitin conjugation, and the Km and Vmax for these reactions are both higher than for the wild type protein. The ability of E3 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 E3 that bind destabilizing N-terminal amino acids in type I and II substrates also interact with the 3C proteases.