In order to study in detail the generality of the NUX (N = A, U, G, or C; X = A, U, or C) rule for the GUC triplet adjacent to the cleavage site in hammerhead ribozymes, two kinetic parameters, namely, kcat and Km, were determined for substrates with mutations in this triplet, which included double mutants with mutations of both N and X. All substrates with mutated cleavage sites were cleaved with reduced efficiency compared to the wild type. However, some mutations mainly affected kcat and others mainly affected Km, a phenomenon that could not have been predicted from previous results. A as the first or third base increased Km by 35- or 30-fold, respectively, while the effect on kcat was small. U as the first or third base decreased kcat by 8- or 15-fold, respectively, while the effect on Km was small. The effect of C as the first base on kinetic parameters was relatively small. The kinetic parameters of double mutants generally were determined by the effects of both individual point mutations. The AUA triplet gave a very much higher kcat than the other double mutants tested. In general, all of the mutants except for the mutant substrate with the CUC triplet had very low cleavage efficiencies, which ranged from 0.6% to 8% of the wild-type value, as a result of the deleterious effects of the mutations on kcat, Km, or both.(ABSTRACT TRUNCATED AT 250 WORDS)