High throughput and quantitative enzymology in the genomic era.

Accurate predictions from models based on physical principles are the ultimate metric of our biophysical understanding. Although there has been stunning progress toward structure prediction, quantitative prediction of enzyme function has remained challenging. Realizing this goal will require large numbers of quantitative measurements of rate and binding constants and the use of these ground-truth data sets to guide the development and testing of these quantitative models. Ground truth data more closely linked to the underlying physical forces are also desired. Here, we describe technological advances that enable both types of ground truth measurements. These advances allow classic models to be tested, provide novel mechanistic insights, and place us on the path toward a predictive understanding of enzyme structure and function.

Revealing enzyme functional architecture via high-throughput microfluidic enzyme kinetics.

Systematic and extensive investigation of enzymes is needed to understand their extraordinary efficiency and meet current challenges in medicine and engineering. We present HT-MEK (High-Throughput Microfluidic Enzyme Kinetics), a microfluidic platform for high-throughput expression, purification, and characterization of more than 1500 enzyme variants per experiment. For 1036 mutants of the alkaline phosphatase PafA (phosphate-irrepressible alkaline phosphatase of Flavobacterium), we performed more than 670,000 reactions and determined more than 5000 kinetic and physical constants for multiple substrates and inhibitors. We uncovered extensive kinetic partitioning to a misfolded state and isolated catalytic effects, revealing spatially contiguous regions of residues linked to particular aspects of function. Regions included active-site proximal residues but extended to the enzyme surface, providing a map of underlying architecture not possible to derive from existing approaches. HT-MEK has applications that range from understanding molecular mechanisms to medicine, engineering, and design.

Protein enhancement of hammerhead ribozyme catalysis.

When the recognition sequence of a ribozyme is extended beyond a certain length, turnover is slowed and specificity is decreased. Here, it is shown that a protein can help a ribozyme overcome these general limitations on ribozyme activity. Cleavage of an RNA oligonucleotide by a hammerhead ribozyme is enhanced 10- to 20-fold upon addition of a protein derived from the p7 nucleocapsid (NC) protein of human immunodeficiency virus-type 1. The NC protein also enhances the ability of the ribozyme to discriminate between cleavage of RNA oligonucleotides with differing sequences. These catalytic improvements can be attributed to the strand exchange activity of this RNA binding protein. It is conceivable that endogenous or added proteins may provide analogous increases in ribozyme activity and specificity in vivo.

The energetics of hydrogen bonds in model systems: implications for enzymatic catalysis.

Low-barrier or short, strong hydrogen bonds have been proposed to contribute 10 to 20 kilocalories per mole to transition-state stabilization in enzymatic catalysis. The proposal invokes a large increase in hydrogen bond energy when the pKa values of the donor and acceptor (where Ka is the acid constant) become matched in the transition state (delta pKa=0). This hypothesis was tested by investigating the energetics of hydrogen bonds as a function of delta pKa for homologous series of compounds under nonaqueous conditions that are conducive to the formation of low-barrier hydrogen bonds. In all cases, there was a linear correlation between the increase in hydrogen-bond energy and the decrease in delta pKa, as expected from simple electrostatic effects. However, no additional energetic contribution to the hydrogen bond was observed at delta pKa=0. These results and those of other model studies suggest alternative mechanisms by which hydrogen bonds can contribute to enzymatic catalysis, in accord with conventional electrostatic considerations.

Role of 4.5S RNA in assembly of the bacterial signal recognition particle with its receptor.

The mechanism by which a signal recognition particle (SRP) and its receptor mediate protein targeting to the endoplasmic reticulum or to the bacterial plasma membrane is evolutionarily conserved. In Escherichia coli, this reaction is mediated by the Ffh/4.5S RNA ribonucleoprotein complex (Ffh/4.5S RNP; the SRP) and the FtsY protein (the SRP receptor). We have quantified the effects of 4.5S RNA on Ffh-FtsY complex formation by monitoring changes in tryptophan fluorescence. Surprisingly, 4.5S RNA facilitates both assembly and disassembly of the Ffh-FtsY complex to a similar extent. These results provide an example of an RNA molecule facilitating protein-protein interactions in a catalytic fashion.

A single-molecule study of RNA catalysis and folding.

Using fluorescence microscopy, we studied the catalysis by and folding of individual Tetrahymena thermophila ribozyme molecules. The dye-labeled and surface-immobilized ribozymes used were shown to be functionally indistinguishable from the unmodified free ribozyme in solution. A reversible local folding step in which a duplex docks and undocks from the ribozyme core was observed directly in single-molecule time trajectories, allowing the determination of the rate constants and characterization of the transition state. A rarely populated docked state, not measurable by ensemble methods, was observed. In the overall folding process, intermediate folding states and multiple folding pathways were observed. In addition to observing previously established folding pathways, a pathway with an observed folding rate constant of 1 per second was discovered. These results establish single-molecule fluorescence as a powerful tool for examining RNA folding.

Probing the folding landscape of the Tetrahymena ribozyme: commitment to form the native conformation is late in the folding pathway.

Large, structured RNAs traverse folding landscapes in which intermediates and long-lived misfolded states are common. To obtain a comprehensive description of the folding landscape for a structured RNA, it is necessary to understand the connections between productive folding pathways and pathways to these misfolded states. The Tetrahymena group I ribozyme partitions between folding to the native state and to a long-lived misfolded conformation. Here, we show that the observed rate constant for commitment to fold to the native or misfolded states is 1.9 min(-1) (37 degrees C, 10 mM Mg(2+)), the same within error as the rate constant for overall folding to the native state. Thus, the commitment to alternative folding pathways is made late in the folding process, concomitant with or after the rate-limiting step for overall folding. The ribozyme forms much of its tertiary structure significantly faster than it reaches this commitment point and the tertiary structure is expected to be stable, suggesting that the commitment to fold along pathways to the native or misfolded states is made from a partially structured intermediate. These results allow the misfolded conformation to be incorporated into a folding framework that reconciles previous data and gives quantitative information about the energetic topology of the folding landscape for this RNA.

New pathways in folding of the Tetrahymena group I RNA enzyme.

Previous studies have shown that the earliest detectable step in folding of the Tetrahymena ribozyme is tertiary structure formation of the peripheral element P5abc. This, along with other results, has suggested that P5abc may serve as a scaffold upon which additional tertiary structure is built. Herein we use the onset of oligonucleotide cleavage activity as a readout for native state formation and investigate the effect of P5abc on the rate of folding to the native structure. Despite the early folding of P5abc, its removal to give the E delta P5abc variant decreases the rate of attainment of an active structure less than fivefold (20-100 mM Mg2+, 15-50 degrees C). Furthermore, P5abc added in trans is able to bind the folded E delta P5abc ribozyme and promote oligonucleotide cleavage at least tenfold more rapidly than folding of the wild-type ribozyme, indicating that E delta P5abc does not have to first unfold before productively binding P5abc to form the true native state. This suggests that a state with the overall tertiary structure formed but with P5abc unfolded represents a viable on-pathway intermediate for the wild-type ribozyme. These results provide strong evidence for the existence of two pathways to the native state: in one pathway P5abc forms tertiary structure first, and in another it forms late. The pathway in which P5abc forms first is favored because P5abc can fold quickly and because its tertiary structure is stable in the absence of additional structured elements, not because P5abc formation is required for subsequent folding steps. In the course of these experiments, we also found that most of the ribozyme population does not reach the native state directly under standard conditions in vitro, but instead forms an inactive structure that is stable for hours. Finally, the fraction that does fold to the native state folds with a single rate constant of 1 min-1, suggesting that there are no significantly populated "fast-track" pathways that reach the native state directly by avoiding slow folding steps.

Mapping the transition state for ATP hydrolysis: implications for enzymatic catalysis.

BACKGROUND: Phosphoryl transfer, typically involving high energy phosphate donors such as ATP, is the most common class of biological reactions. Despite this, the transition state for phosphoryl transfer from ATP in solution has not been systematically investigated. Characterization of the transition state for the uncatalyzed hydrolysis of ATP would provide a starting point for dissection of enzyme-catalyzed reactions. RESULTS: We examined phosphoryl transfer from ATP, GTP and pyrophosphate to a series of alcohols; these reactions are analogous to the phosphorylation of sugars and other biological alcohols and to the hydrolysis of ATP. The Brønsted beta(nucleophile) value of 0.07 is small, indicating that there is little bond formation between the incoming nucleophile and the electrophilic phosphoryl group in the transition state. Coordination of Mg2+ has no measurable effect on this value. The Brønsted beta(leaving group) value of -1.1 for phosphoryl transfer to water from a series of phosphoanhydrides is large and negative, suggesting that the bond between phosphorous and the leaving group oxygen is largely broken in the transition state. CONCLUSIONS: Uncatalyzed hydrolysis of ATP in solution occurs via a dissociative, metaphosphate-like transition state, with little bond formation between nucleophile and ATP and substantial cleavage of the bond between the gamma-phosphoryl moiety and the ADP leaving group. Bound Mg2+ does not perturb the dissociative nature of the transition state, contrary to proposals that enzyme-bound metal ions alter this structure. The simplest expectation for phosphoryl transfer at the active site of enzymes thus entails a dissociative transition state. These results provide a basis for analyzing catalytic mechanisms for phosphoryl transfer.

Catalytic promiscuity and the evolution of new enzymatic activities.

Several contemporary enzymes catalyze alternative reactions distinct from their normal biological reactions. In some cases the alternative reaction is similar to a reaction that is efficiently catalyzed by an evolutionary related enzyme. Alternative activities could have played an important role in the diversification of enzymes by providing a duplicated gene a head start towards being captured by adaptive evolution.

The role of the cleavage site 2'-hydroxyl in the Tetrahymena group I ribozyme reaction.

BACKGROUND: The 2'-hydroxyl of U preceding the cleavage site, U(-1), in the Tetrahymena ribozyme reaction contributes 10(3)-fold to catalysis relative to a 2'-hydrogen atom. Previously proposed models for the catalytic role of this 2'-OH involve coordination of a catalytic metal ion and hydrogen-bond donation to the 3'-bridging oxygen. An additional model, hydrogen-bond donation by the 2'-OH to a nonbridging reactive phosphoryl oxygen, is also consistent with previous results. We have tested these models using atomic-level substrate modifications and kinetic and thermodynamic analyses. RESULTS: Replacing the 2'-OH with -NH(3)(+) increases the reaction rate approximately 60-fold, despite the absence of lone-pair electrons on the 2'-NH(3)(+) group to coordinate a metal ion. Binding and reaction of a modified oligonucleotide substrate with 2'-NH(2) at U(-1) are unaffected by soft-metal ions. These results suggest that the 2'-OH of U(-1) does not interact with a metal ion. The contribution of the 2'-moiety of U(-1) is unperturbed by thio substitution at either of the nonbridging oxygens of the reactive phosphoryl group, providing no indication of a hydrogen bond between the 2'-OH and the nonbridging phosphoryl oxygens. In contrast, the 10(3)-fold catalytic advantage of 2'-OH relative to 2'-H is eliminated when the 3'-bridging oxygen is replaced by sulfur. As sulfur is a weaker hydrogen-bond acceptor than oxygen, this effect suggests a hydrogen-bonding interaction between the 2'-OH and the 3'-bridging oxygen. CONCLUSIONS: These results provide the first experimental support for the model in which the 2'-OH of U(-1) donates a hydrogen bond to the neighboring 3'-bridging oxygen, thereby stabilizing the developing negative charge on the 3'-oxygen in the transition state.

Hydrogen bonding in enzymatic catalysis: analysis of energetic contributions.

Enzymes can provide catalysis by increasing the strengthening of hydrogen bonds to groups undergoing charge rearrangement in the course of reaction relative to the strengthening of the hydrogen bonds in the corresponding solution reactions. This can be accomplished by using hydrogen bond donors and acceptors that are stronger than water and by lowering the effective dielectric relative to that in aqueous solution. We suggest that these electrostatic effects are of general significance in enzymatic catalysis. The effective dielectric is lowered by the overall "rigidity" of the folded enzyme, which facilitates the formation of active site interactions, and by the fixation of active site functional groups within the enzyme x substrate complex. This underscores the fundamental interconnection of catalytic mechanisms in enzymatic catalysis.

Evidence for processivity and two-step binding of the RNA substrate from studies of J1/2 mutants of the Tetrahymena ribozyme.

J1/2 of the Tetrahymena ribozyme, a sequence of three A residues, connects the RNA-binding site to the catalytic core. Addition or deletion of bases from J1/2 improves turnover and substrate specificity in the site-specific endonuclease reaction catalyzed by this ribozyme: G2CCCUCUA5 (S) + G in-equilibrium G2CCCUCU (P) + GA5. These paradoxical enhancements are caused by decreased affinity of the ribozyme for S and P [Young, B., Herschlag, D., & Cech, T.R. (1991) Cell 67, 1007]. An additional property of these mutant ribozymes, decreased fidelity of RNA cleavage, is now analyzed. (Fidelity is the ability to cleave at the correct phosphodiester bond within a particular RNA substrate.) Introduction of deoxy residues to give "chimeric" ribo/deoxyribooligonucleotides changes the positions of incorrect cleavage. Previous work indicated that S is bound to the ribozyme by both base pairing and teritary interactions involving 2'-hydroxyl groups of S. The data herein strongly suggest that the P1 duplex, which consists of S base-paired with the 5' exon binding site of the ribozyme, can dock into tertiary interactions in different registers; different 2'-hydroxyl groups of S plug into tertiary contacts with the ribozyme in the different registers. It is concluded that the mutations decrease fidelity by increasing the probability of docking out of register relative to docking in the normal register, thereby giving cleavage at different positions along S. These data also show that the contribution of J1/2 to the teritiary interactions is indirect, not direct. Thus, a structural role of the nonconserved J1/2 is indicated: this sequence positions S to optimize tertiary binding interactions and to ensure cleavage at the phosphodiester bond corresponding to the 5' splice site. Substitution of sulfur for the nonbridging pro-RP oxygen atom at the normal cleavage site has no effect on (kcat/Km)S but decreases the fraction of cleavage at the normal site in reactions catalyzed by the -3A mutant ribozyme, which has all three A residues of J1/2 removed. Thus, the ribozyme chooses where to cleave S after rate-limiting binding of S, indicating that docking can change after binding and suggesting that the ribozyme could act processively. Indeed, it is shown that the +2A ribozyme cleaves at one position along an RNA substrate and then, before releasing that RNA product, cleaves it again.(ABSTRACT TRUNCATED AT 400 WORDS)

Implications of ribozyme kinetics for targeting the cleavage of specific RNA molecules in vivo: more isn't always better.

Kinetic and thermodynamic factors that determine specificity of RNA cleavage by ribozymes are illustrated with examples from recent work with a ribozyme derived from the group I intron of Tetrahymena thermophila pre-rRNA. The conclusions also apply to other ribozymes, to antisense oligonucleotide experiments, and to RNA and DNA cleavage agents that can recognize a single-stranded or double-stranded region of variable length. At first, adding bases to a ribozyme's recognition sequence is expected to increase cleavage of the target RNA relative to cleavage of other RNAs. However, adding more bases ultimately reduces this discrimination, as cleavage occurs essentially every time the target RNA or a mismatched RNA binds the ribozyme. This occurs despite the weaker binding of the mismatched RNA because dissociation becomes too slow (binding is too strong) to allow the ribozyme to "choose" between cleavage of the target RNA and a mismatched RNA. In summary, more (base pairing) isn't always better, because maximal discrimination requires equilibrium binding prior to cleavage. The maximum discrimination that can be obtained is expected to be greater with an A + U-rich recognition sequence than with a G + C-rich recognition sequence. This is because the weaker A.U base pairs (relative to G-C base pairs) allow recognition to be spread over a larger number of bases while preventing binding that is too strong. Finally, creating an A-rich ribozyme rather than a U-rich ribozyme avoids the loss in discrimination expected with U-rich ribozymes from the formation of U.G wobble pairs in addition to the "targeted" Watson-Crick U.A pair.

Comparison of pH dependencies of the Tetrahymena ribozyme reactions with RNA 2'-substituted and phosphorothioate substrates reveals a rate-limiting conformational step.

The L-21 ScaI ribozyme (E) derived from the self-splicing group I intron of Tetrahymena pre-rRNA catalyzes an RNA endonuclease reaction analogous to the first step in self-splicing: CCCUCUAAAAA (S) + G-->CCCUCU+GAAAAA. We show herein that the pH dependence for the single-turnover reaction E.S+G-->products follows a pH dependence with pKapp = 6.9 (10 mM MgCl2, 50 degrees C). This result was surprising because the titratable groups of RNA have pKa values of < approximately 4 or > approximately 9. Thus, two models were considered: (i) the ribozyme structure perturbs a pKa such that the pKapp of 6.9 corresponds to an actual titration or (ii) the pKapp is a kinetic pKa, reflecting a change in the rate-limiting step rather than an actual titration. Oligonucleotide substrates with -H (deoxyribose), -F (2'-fluoro-2'-deoxyribose), and -OH (ribose) substitutions at the 2' position of the U residue at the cleavage site [U(-1)] vary considerably in their intrinsic reactivities. In the ribozyme reaction these substrates reacted at very different rates at low pH, but approached the same limiting reaction rate at high pH. Similarly, substitution of the pro-RP nonbridging oxygen atom of the reactive phosphoryl group by sulfur lowers the intrinsic reactivity of the oligonucleotide substrate. In the ribozyme reaction, this "thio effect" was 2.3 below pH 6.9, whereas the thio substitution had no effect on the rate above pH 6.9.(ABSTRACT TRUNCATED AT 250 WORDS)

The nature of the transition state for enzyme-catalyzed phosphoryl transfer. Hydrolysis of O-aryl phosphorothioates by alkaline phosphatase.

There has been much speculation that enzymes change the nature of the transition state for phosphoryl transfer from the dissociative transition state observed in solution reactions to an associative transition state at the enzyme's active site. This proposal can be tested by comparing linear free energy relationships (LFERs) for nonenzymatic and enzymatic reactions, provided that the specificity of the enzyme's binding site does not perturb the dependence of rate on the intrinsic reactivity of a series of substrates. The shallow binding groove of Escherichia coli alkaline phosphatase (AP) and its wide specificity suggest that this enzyme may be suited for such an approach. A second requirement of this approach is that the actual chemical step is rate-limiting. Comparisons of the reactions of aryl phosphorothioates and aryl phosphates support the previous conclusion that a nonchemical step limits kcat/KM for reactions of aryl phosphates, but suggest that the chemical cleavage step is rate-limiting for the aryl phosphorothioates. We therefore determined the dependence of the rate of AP-catalyzed cleavage of a series of aryl phosphorothioates on the intrinsic reactivity of the substrates. The large negative values of beta leaving group = -0.8 for the enzymatic reaction (kcat/KM) and -1.1 for the nonenzymatic hydrolysis reaction suggest that there is considerable dissociative character in both the enzymatic and nonenzymatic transition states. Despite the wide specificity of AP, certain substrates deviate from the LFER, underscoring that extreme care is required in applying LFERs to enzymatic reactions. The large negative value of beta leaving group suggests that AP can achieve substantial catalysis via a transition state with dissociative character.

Mechanistic investigations of a ribozyme derived from the Tetrahymena group I intron: insights into catalysis and the second step of self-splicing.

Self-splicing of Tetrahymena pre-rRNA proceeds in two consecutive phosphoryl transesterification steps. One major difference between these steps is that in the first an exogenous guanosine (G) binds to the active site, while in the second the 3'-terminal G414 residue of the intron binds. The first step has been extensively characterized in studies of the L-21ScaI ribozyme, which uses exogenous G as a nucleophile. In this study, mechanistic features involved in the second step are investigated by using the L-21G414 ribozyme. The L-21G414 reaction has been studied in both directions, with G414 acting as a leaving group in the second step and a nucleophile in its reverse. The rate constant of chemical step is the same with exogenous G bound to the L-21ScaI ribozyme and with the intramolecular guanosine residue of the L-21G414 ribozyme. The result supports the previously proposed single G-binding site model and further suggests that the orientation of the bound G and the overall active site structure is the same in both steps of the splicing reaction. An evolutionary rationale for the use of exogenous G in the first step is also presented. The results suggest that the L-21G414 ribozyme exists predominantly with the 3'-terminal G414 docked into the G-binding site. This docking is destabilized by approximately 100-fold when G414 is attached to an electron-withdrawing pA group. The internal equilibrium with K(int) = 0.7 for the ribozyme reaction indicates that bound substrate and product are thermodynamically matched and is consistent with a degree of symmetry within the active site. These observations are consistent with the presence of a second Mg ion in the active site. Finally, the slow dissociation of a 5' exon analog relative to a ligated exon analog from the L-21G414 ribozyme suggests a kinetic mechanism for ensuring efficient ligation of exons and raises new questions about the overall self-splicing reaction.

Specificity from steric restrictions in the guanosine binding pocket of a group I ribozyme.

The 3' splice site of group I introns is defined, in part, by base pairs between the intron core and residues just upstream of the splice site, referred to as P9.0. We have studied the specificity imparted by P9.0 using the well-characterized L-21 Scal ribozyme from Tetrahymena by adding residues to the 5' end of the guanosine (G) that functions as a nucleophile in the oligonucleotide cleavage reaction: CCCUCUA5 (S) + NNG <--> CCCUCU + NNGA5. UCG, predicted to form two base pairs in P9.0, reacts with a (kcat/KM) value approximately 10-fold greater than G, consistent with previous results. Altering the bases that form P9.0 in both the trinucleotide G analog and the ribozyme affects the specificity in the manner predicted for base-pairing. Strikingly, oligonucleotides incapable of forming P9.0 react approximately 10-fold more slowly than G, for which the mispaired residues are simply absent. The observed specificity is consistent with a model in which the P9.0 site is sterically restricted such that an energetic penalty, not present for G, must be overcome by G analogs with 5' extensions. Shortening S to include only one residue 3' of the cleavage site (CCCUCUA) eliminates this penalty and uniformly enhances the reactions of matched and mismatched oligonucleotides relative to guanosine. These results suggest that the 3' portion of S occupies the P9.0 site, sterically interfering with binding of G analogs with 5' extensions. Similar steric effects may more generally allow structured RNAs to avoid formation of incorrect contacts, thereby helping to avoid kinetic traps during folding and enhancing cooperative formation of the correct structure.

Catalysis of the hydrolysis of phosphorylated pyridines by Mg(OH)+: a possible model for enzymatic phosphoryl transfer.

The second-order rate constants for reaction of the Mg2+ complexes of phosphorylated pyridine monoanions with Mg(OH)+ are 10(4)-10(6)-fold larger than the second-order rate constants for their reaction with water (25 degrees C, ionic strength 1.5). Of the 10(6)-fold rate enhancement with the phosphorylated 4-morpholinopyridine/Mg2 complex, approximately 10(4)-fold is attributed to the greater nucleophilicity of Mg(OH)+ compared with water. The remaining catalysis of approximately 10(2)-fold is attributed to induced intramolecularity from positioning of the hydroxide ion and phosphoryl group by the Mg2+ ions. This reaction may provide a model for the role of a metal ion in increasing the concentration of the anions of enolpyruvate and serine and holding the nucleophile in the correct position for phosphoryl transfer in the reactions catalyzed by pyruvate kinase and alkaline phosphatase, for example. Some mechanisms that can provide catalysis of phosphoryl transfer through a metaphosphate-like transition state are reviewed briefly.

Catalysis of RNA cleavage by the Tetrahymena thermophila ribozyme. 1. Kinetic description of the reaction of an RNA substrate complementary to the active site.

A ribozyme derived from the intervening sequence (IVS) of the Tetrahymena preribosomal RNA catalyzes a site-specific endonuclease reaction: G2CCCUCUA5 + G in equilibrium with G2CCCUCU + GA5 (G = guanosine). This reaction is analogous to the first step in self-splicing of the pre-rRNA, with the product G2CCCUCU analogous to the 5'-exon. The following mechanistic conclusions have been derived from pre-steady-state and steady-state kinetic measurements at 50 degrees C and neutral pH in the presence of 10 mM Mg2+. The value of kcat/Km = 9 x 10(7) M-1 min-1 for the oligonucleotide substrate with saturating G represents rate-limiting binding. This rate constant for binding is of the order expected for formation of a RNA.RNA duplex between oligonucleotides. (Phylogenetic and mutational analyses have shown that this substrate is recognized by base pairing to a complementary sequence within the IVS). The value of kcat = 0.1 min-1 represents rate-limiting dissociation of the 5'-exon analogue, G2CCCUCU. The product GA5 dissociates first from the ribozyme because of this slow off-rate for G2CCCUCU. The similar binding of the product, G2CCCUCU, and the substrate, G2CCCUCUA5, to the 5'-exon binding site of the ribozyme, with Kd = 1-2 nM, shows that the pA5 portion of the substrate makes no net contribution to binding. Both the substrate and product bind approximately 10(4)-fold (6 kcal/mol) stronger than expected from base pairing with the 5'-exon binding site. Thus, tertiary interactions are involved in binding. Binding of G2CCCUCU and binding of G are independent. These and other data suggest that binding of the oligonucleotide substrate, G2CCCUCUA5, and binding of G are essentially random and independent. The rate constant for reaction of the ternary complex is calculated to be kc approximately equal to 350 min-1, a rate constant that is not reflected in the steady-state rate parameters with saturating G. The simplest interpretation is adopted, in which kc represents the rate of the chemical step. A site-specific endonuclease reaction catalyzed by the Tetrahymena ribozyme in the absence of G was observed; the rate of the chemical step with solvent replacing guanosine, kc(-G) = 0.7 min-1, is approximately 500-fold slower than that with saturating guanosine. The value of kcat/Km = 6 x 10(7) M-1 min-1 for this hydrolysis reaction is only slightly smaller than that with saturating guanosine, because the binding of the oligonucleotide substrate is predominantly rate-limiting in both cases.(ABSTRACT TRUNCATED AT 400 WORDS)