SITE-DIRECTED MUTAGENESIS OF UBIQUITIN - DIFFERENTIAL ROLES FOR ARGININE IN THE INTERACTION WITH UBIQUITIN-ACTIVATING ENZYME

The strict evolutionary conservation of ubiquitin suggests an essential role for each residue in the folding, stability, or function of the protein but precludes identification of such contributions through interspecies comparison of ubiquitin sequences. However, site-directed mutagenesis potentially allows assignment of specific function(s) for each residue. The four arginines present on ubiquitin at positions 42, 54, 72, and 74 were independently mutated to leucine and their effects on the interaction of the resulting polypeptides with ubiquitin-activating enzyme (E1) were characterized. All of the mutants except UbR54L exhibited altered kinetics for E1-catalyzed ATP:PPi exchange compared to wild-type ubiquitin. In addition, the UbR72L mutant altered the mechanism of El from strictly order addition of substrates to random addition with respect to ATP and ubiquitin. Values for the intrinsic K-d of ubiquitin binding were determined by coupling the net forward reaction of E1 to the E2(32K)-catalyzed conjugation of histone H2B. Only R54 and R72 residues participate in the initial binding of free ubiquitin, resulting in a 6- or 58-fold increase in K-d for UbR54L or UbR72L, respectively, compared to wild type. More significant effects of the UbR42L and UbR72L mutants were observed for binding of their respective ubiquitin adenylate intermediates within the E1 active site. Wild-type ubiquitin adenylate binds to E1 with an estimated K-d less than or equal to 8 X 10(-12) M while intermediates formed with UbR42L or UbR72L each bind with ca. 10(3)-fold lower affinity, representing a destabilization of greater than or equal to 7 kcal/mol. In contrast, neither R54 or R74 significantly contribute to ubiquitin adenylate binding since their mutations did not exhibit the same behavior. These results indicate that three of the arginines contribute to the interaction between ubiquitin and E1 but do not quantitatively account for polypeptide binding, indicating roles for other residues. Moreover, the differential contributions of these residues to binding of either free ubiquitin or adenylate and the relative magnitudes of the effects are consistent with a significant reorientation of the active site during the catalytic cycle of E1.