A kinetic mechanism is presented for the cleavage of Bacillus subtilis precursor tRNA(Asp) catalyzed by the RNA component of B. subtilis ribonuclease P (RNase P) under optimal conditions (50 mM Tris Cl (pH 8.0), 100 mM MgCl2, and 800 mM NH4Cl, 37 degrees C). This kinetic mechanism was derived from measuring pre-steady-state, steady-state, single-turnover, and binding kinetics using a combination of quench-flow, gel filtration, and gel shift techniques. A minimal kinetic description involves the following: (1) binding of pre-tRNA(Asp) to RNase P RNA rapidly (6.3 x 10(6) M-1 s-1), but slower than the diffusion-controlled limit; (2) cleavage of the phosphodiester bond with a rate constant of 6 s-1; (3) dissociation of products in a kinetically preferred pathway, with the 5' RNA fragment dissociating first (> or = 0.2 s-1) followed by rate-limiting tRNA dissociation (0.02 s-1); and (4) formation of a second conformer of RNase P RNA during the catalytic cycle that is less stable and binds pre-tRNA(Asp) significantly more slowly (7 x 10(4) M-1 s-1). This scheme involves the isolation of individual steps in the reaction sequence, is consistent with steady-state data, and pinpoints the rate-determining step under a variety of conditions. This kinetic mechanism will facilitate a more accurate definition of the role of metals, pH, and the protein component in each step of the reaction and provide an essential background for understanding the influence of structural changes on the catalytic activity.

The multiple roles Mg2+ plays in ribozyme-catalyzed reactions in stabilizing RNA structure, enhancing the affinity of bound substrates, and increasing catalysis are delineated for the RNA component of ribonuclease P (RNase P RNA) by a combination of steady-state kinetics, transient kinetics, and equilibrium binding measurements. Divalent metal ions cooperatively increase the affinity of Bacillus subtilis RNase P RNA for B. subtilis tRNA(Asp) more than 10(3)-fold, consistent with at least two additional magnesium ions binding to the RNase P RNA.tRNA complex. Monovalent cations also decrease KD(tRNA) and reduce, but do not eliminate, the dependence on magnesium ions, demonstrating that nonspecific electrostatic shielding is not sufficient to explain the requirement for high salt. Both di- and monovalent cations promote the high affinity of tRNA by forming contacts in the binary complex that reduce the dissociation rate constant for tRNA. Additionally, the hyperbolic dependence of the hydrolytic rate constant on the concentration of magnesium with a K1/2 approximately equal to 36 mM suggests that a third low-affinity divalent metal ion stabilizes the transition state for pre-tRNA cleavage. Furthermore, many (about 100) magnesium ions bind independently to RNase P RNA with higher affinity than the K1/2 of any of the functionally characterized magnesium binding sites. Therefore, the magnesium binding sites that have differential affinity in either the "folded" species or binary complex are a small subset of the total number of associated magnesium ions. In summary, the importance of magnesium bound to RNase P RNA can be separated functionally into three crucial roles: at least three sites stabilize the folded RNA tertiary structure [Pan. T. (1995) Biochemistry 34, 902-909], at least two sites enhance the formation of complexes of RNase P RNA with pre-tRNA or tRNA, and at least one site stabilizes the transition state for pre-tRNA cleavage.

Galactose mutarotase catalyzes the interconversion of alpha- and beta-anomers of aldoses and is a recently identified member of the gal operon of Escherichia coli and participant in the Leloir pathway [Bouffard et al. (1994) J. Mol. Biol. 244, 269-278]. We report the purification and characterization of this enzyme, as well as mechanistic studies involving chemical modification with diethylpyrocarbonate (DEPC) and site-directed mutagenesis demonstrating the significance of two conserved histidine residues. The enzyme lacks metal ions and oxidoreduction cofactors, and an extinction coefficient of (6.2 +/- 0.4) x 10(4) M-1 cm-1 has been measured by quantitative amino acid analysis. The catalytic mechanism is likely concerted general acid/general base. Experiments involving modification with DEPC suggest that a histidine is essential and is protected by substrate. Furthermore, site-directed mutagenesis of two conserved histidines was performed, and characterization of these mutants (His104Gln and His175Asn) illustrates the significance of these residues. Kinetic analysis of H104Q demonstrates an increase in KM of about 600-fold, a decrease in kcat of approximately 7-fold, and a 4000-fold decrease in kcat/KM as compared to the wild-type enzyme. The activity of His175Asn mutant, on the other hand, was too low to be measured accurately, and His 175 remains a candidate for the general base. These mutants were also subjected to DEPC modification, and results are consistent with the presence of two important histidines positioned closely together in the active site.