tRNase Z, which can endonucleolytically remove pre-tRNA 39-end trailers, possesses the signature His domain (HxHxDH; Motif II) of the b-lactamase family of metal-dependent hydrolases. Motif II combines with Motifs III-V on its carboxy side to coordinate two divalent metal ions, constituting the catalytic core. The PxKxRN loop and Motif I on the amino side of Motif II have been suggested to modulate tRNase Z activity, including the anti-determinant effect of CCA in mature tRNA. Ala walks through these two homology blocks reveal residues in which the substitutions unexpectedly reduce catalytic efficiency. While substitutions in Motif II can drastically affect k cat without affecting k M , five-to 15-fold increases in k M are observed with substitutions in several conserved residues in the PxKxRN loop and Motif I. These increases in k M suggest a model for substrate binding. Expressed tRNase Z processes mature tRNA with CCA at the 39 end ;80 times less efficiently than a pre-tRNA possessing natural sequence of the 39-end trailer, due to reduced k cat with no effect on k M , showing the CCA anti-determinant to be a characteristic of this enzyme.