Class I aminoacyl-tRNA synthetases catalyze editing reactions that prevent ambiguity from entering the genetic code. Misactivated amino acids are translocated in cis from the active site for aminoacylation to the center for editing, located approximately 30 A away. Mutational analysis has functionally separated the two sites by creating mutations that disrupt the catalytic center for editing but not for aminoacylation and vice versa. What is not known is whether translocation per se can be disrupted without an effect on either catalytic center. Here we describe mutations in a presumptive "hinge region" of isoleucyl-tRNA synthetase that is situated between the two sites. Interstice mutations had little or no effect on either catalytic center. In contrast, the same specific mutations disrupted translocation. Thus, with these mutations all three functions, translocation, catalysis of aminoacylation, and editing, have been mutationally separated. The results are consistent with translocation involving a hinge-region conformational shift that does not perturb the two catalytic centers.