Discovery of MK-1454: A Potent Cyclic Dinucleotide Stimulator of Interferon Genes Agonist for the Treatment of Cancer.

Stereochemically and structurally complex cyclic dinucleotide-based stimulator of interferon genes (STING) agonists were designed and synthesized to access a previously unexplored chemical space. The assessment of biochemical affinity and cellular potency, along with computational, structural, and biophysical characterization, was applied to influence the design and optimization of novel STING agonists, resulting in the discovery of MK-1454 as a molecule with appropriate properties for clinical development. When administered intratumorally to immune-competent mice-bearing syngeneic tumors, MK-1454 exhibited robust tumor cytokine upregulation and effective antitumor activity. Tumor shrinkage in mouse models that are intrinsically resistant to single-agent therapy was further enhanced when treating the animals with MK-1454 in combination with a fully murinized antimouse PD-1 antibody, mDX400. These data support the development of STING agonists in combination with pembrolizumab (humanized anti-PD-1 antibody) for patients with tumors that are partially responsive or nonresponsive to single-agent anti-PD-1 therapy.

An orally available non-nucleotide STING agonist with antitumor activity.

Pharmacological activation of the STING (stimulator of interferon genes)-controlled innate immune pathway is a promising therapeutic strategy for cancer. Here we report the identification of MSA-2, an orally available non-nucleotide human STING agonist. In syngeneic mouse tumor models, subcutaneous and oral MSA-2 regimens were well tolerated and stimulated interferon-ß secretion in tumors, induced tumor regression with durable antitumor immunity, and synergized with anti-PD-1 therapy. Experimental and theoretical analyses showed that MSA-2 exists as interconverting monomers and dimers in solution, but only dimers bind and activate STING. This model was validated by using synthetic covalent MSA-2 dimers, which were potent agonists. Cellular potency of MSA-2 increased upon extracellular acidification, which mimics the tumor microenvironment. These properties appear to underpin the favorable activity and tolerability profiles of effective systemic administration of MSA-2.

Discovery and optimization of heteroaryl piperazines as potent and selective PI3Kδ inhibitors.

A high-throughput screening (HTS) campaign identified a class of heteroaryl piperazines with excellent baseline affinity and selectivity for phosphoinositide 3-kinase δ (PI3Kδ) over closely related isoforms. Rapid evaluation and optimization of structure-activity relationships (SAR) for this class, leveraging the modular nature of this scaffold, facilitated development of this hit class into a series of potent and selective inhibitors of PI3Kδ. This effort culminated in the identification of 29, which displayed excellent potency in enzyme and cell-based assays, as well as favorable pharmacokinetic and off-target profiles.

Discovery of a Novel cGAMP Competitive Ligand of the Inactive Form of STING.

Drugging large protein pockets is a challenge due to the need for higher molecular weight ligands, which generally possess undesirable physicochemical properties. In this communication, we highlight a strategy leveraging small molecule active site dimers to inhibit the large symmetric binding pocket in the STING protein. By taking advantage of the 2:1 binding stoichiometry, maximal buried interaction with STING protein can be achieved while maintaining the ligand physicochemical properties necessary for oral exposure. This mode of binding requires unique considerations for potency optimization including simultaneous optimization of protein-ligand as well as ligand-ligand interactions. Successful implementation of this strategy led to the identification of 18, which exhibits good oral exposure, slow binding kinetics, and functional inhibition of STING-mediated cytokine release.

Dissecting Therapeutic Resistance to ERK Inhibition.

The MAPK pathway is frequently activated in many human cancers, particularly melanomas. A single-nucleotide mutation in BRAF resulting in the substitution of glutamic acid for valine (V(600E)) causes constitutive activation of the downstream MAPK pathway. Selective BRAF and MEK inhibitor therapies have demonstrated remarkable antitumor responses in BRAF(V600) (E)-mutant melanoma patients. However, initial tumor shrinkage is transient and the vast majority of patients develop resistance. We previously reported that SCH772984, an ERK 1/2 inhibitor, effectively suppressed MAPK pathway signaling and cell proliferation in BRAF, MEK, and concurrent BRAF/MEK inhibitor-resistant tumor models. ERK inhibitors are currently being evaluated in clinical trials and, in anticipation of the likelihood of clinical resistance, we sought to prospectively model acquired resistance to SCH772984. Our data show that long-term exposure of cells to SCH772984 leads to acquired resistance, attributable to a mutation of glycine to aspartic acid (G(186D)) in the DFG motif of ERK1. Structural and biophysical studies demonstrated specific defects in SCH772984 binding to mutant ERK. Taken together, these studies describe the interaction of SCH772984 with ERK and identify a novel mechanism of ERK inhibitor resistance through mutation of a single residue within the DFG motif. Mol Cancer Ther; 15(4); 548-59. ©2016 AACR.

Reactions of Cg10062, a cis-3-Chloroacrylic Acid Dehalogenase Homologue, with Acetylene and Allene Substrates: Evidence for a Hydration-Dependent Decarboxylation.

Cg10062 is a cis-3-chloroacrylic acid dehalogenase (cis-CaaD) homologue from Corynebacterium glutamicum with an unknown function and an uninformative genomic context. It shares 53% pairwise sequence similarity with cis-CaaD including the six active site amino acids (Pro-1, His-28, Arg-70, Arg-73, Tyr-103, and Glu-114) that are critical for cis-CaaD activity. However, Cg10062 is a poor cis-CaaD: it lacks catalytic efficiency and isomer specificity. Two acetylene compounds (propiolate and 2-butynoate) and an allene compound, 2,3-butadienoate, were investigated as potential substrates. Cg10062 functions as a hydratase/decarboxylase using propiolate as well as the cis-3-chloro- and 3-bromoacrylates, generating mixtures of malonate semialdehyde and acetaldehyde. The two activities occur sequentially at the active site using the initial substrate. With 2,3-butadienoate and 2-butynoate, Cg10062 functions as a hydratase and converts both to acetoacetate. Mutations of the proposed water-activating residues (E114Q, E114D, and Y103F) have a range of consequences from a reduction in wild type activity to a switch of activities (i.e., hydratase into a hydratase/decarboxylase or vice versa). The intermediates for the hydration and decarboxylation products can be trapped as covalent adducts to Pro-1 when NaCNBH3 is incubated with the E114D mutant and 2,3-butadienoate or 2-butynoate, and the Y103F mutant and 2-butynoate. Three mechanisms are presented to explain these findings. One mechanism involves the direct attack of water on the substrate, whereas the other two mechanisms use covalent catalysis in which a covalent bond forms between Pro-1 and the hydration product or the substrate. The strengths and weaknesses of the mechanisms and the implications for Cg10062 function are discussed.

A mutational analysis of the active site loop residues in cis-3-Chloroacrylic acid dehalogenase.

cis-3-Chloroacrylic acid dehalogenase (cis-CaaD) from Pseudomonas pavonaceae 170 and a homologue from Corynebacterium glutamicum designated Cg10062 are 34% identical in sequence (54% similar). The former catalyzes a key step in a bacterial catabolic pathway for the nematocide 1,3-dichloropropene, whereas the latter has no known biological activity. Although Cg10062 has the six active site residues (Pro-1, His-28, Arg-70, Arg-73, Tyr-103, and Glu-114) that are critical for cis-CaaD activity, it shows only a low level cis-CaaD activity and lacks the specificity of cis-CaaD: Cg10062 processes both isomers of 3-chloroacrylate with a preference for the cis isomer. The basis for these differences is unknown, but a comparison of the crystal structures of the enzymes covalently modified by an adduct resulting from their incubation with the same inhibitor offers a possible explanation. A six-residue active site loop in cis-CaaD shows a conformation strikingly different from that observed in Cg10062: the loop closes down on the active site of cis-CaaD, but not on that of Cg10062. To examine what this loop might contribute to cis-CaaD catalysis and specificity, the residues were changed individually to those found in Cg10062. Subsequent kinetic and mechanistic analysis suggests that the T34A mutant of cis-CaaD is more Cg10062-like. The mutant enzyme shows a 4-fold increase in Km (using cis-3-bromoacrylate), but not to the degree observed for Cg10062 (687-fold). The mutation also causes a 4-fold decrease in the burst rate (compared to that of wild-type cis-CaaD), whereas Cg10062 shows no burst rate. More telling is the reaction of the T34A mutant of cis-CaaD with the alternate substrate, 2,3-butadienoate. In the presence of NaBH4 and the allene, cis-CaaD is completely inactivated after one turnover because of the covalent modification of Pro-1. The same experiment with Cg10062 does not result in the covalent modification of Pro-1. The different outcomes are attributed to covalent catalysis (using Pro-1) followed by hydrolysis of the enamine or imine tautomer in cis-CaaD versus direct hydration of the allene to yield acetoacetate in the case of Cg10062. The T34A mutant shows partial inactivation, requiring five turnovers of the substrate per monomer, which suggests that the direct hydration route is favored 80% of the time. However, the mutation does not alter the stereochemistry at C-2 of [2-D]acetoacetate when the reaction is conducted in D2O. Both cis-CaaD and the T34 mutant generate (2R)-[2-D]acetoacetate, whereas Cg10062 generates mostly the 2S isomer. The combined observations are consistent with a role for the loop region in cis-CaaD specificity and catalysis, but the precise role remains to be determined.

A pre-steady state kinetic analysis of the αY60W mutant of trans-3-chloroacrylic acid dehalogenase: implications for the mechanism of the wild-type enzyme.

The bacterial degradation of the nematicide 1,3-dichloropropene, an isomeric mixture, requires the action of trans- and cis-3-chloroacrylic acid dehalogenase (CaaD and cis-CaaD, respectively). Both enzymes are tautomerase superfamily members and share a core catalytic mechanism for the hydrolytic dehalogenation of the respective isomer of 3-haloacrylate. The observation that cis-CaaD requires two additional residues raises the question of how CaaD conducts a comparable reaction with fewer catalytic residues. As part of an effort to determine the basis for the apparently simpler CaaD-catalyzed reaction, the kinetic mechanism was determined by stopped-flow and chemical-quench techniques using a fluorescent mutant form of the enzyme, αY60W-CaaD, and trans-3-bromoacrylate as the substrate. The data from these experiments as well as bromide inhibition studies are best accommodated by a six-step model that provides individual rate constants for substrate binding, chemistry, and a proposed conformational change occurring after chemistry followed by release of malonate semialdehyde and bromide. The conformational change and product release rates are comparable, and together they limit the rate of turnover. The kinetic analysis and modeling studies validate the αY60W-CaaD mutant as an accurate reporter of active site events during the course of the enzyme-catalyzed reaction. The kinetic mechanism for the αY60W-CaaD-catalyzed reaction is comparable to that obtained for the cis-CaaD-catalyzed reaction. The kinetic model and the validated αY60W-CaaD mutant set the stage for an analysis of active site mutants to explore the contributions of individual catalytic residues and the basis for the simplicity of the reaction.

Flight of a cytidine deaminase complex with an imperfect transition state analogue inhibitor: mass spectrometric evidence for the presence of a trapped water molecule.

Cytidine deaminase (CDA) binds the inhibitor zebularine as its 3,4-hydrate (K(d) ~ 10(-12) M), capturing all but ~5.6 kcal/mol of the free energy of binding expected of an ideal transition state analogue (K(tx) ~ 10(-16) M). On the basis of its entropic origin, that shortfall was tentatively ascribed to the trapping of a water molecule in the enzyme-inhibitor complex, as had been observed earlier for product uridine [Snider, M. J., and Wolfenden, R. (2001) Biochemistry 40, 11364-11371]. Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS) of CDA nebularized in the presence of saturating 5-fluorozebularine reveals peaks corresponding to the masses of E(2)Zn(2)W(2) (dimeric Zn-CDA with two water molecules), E(2)Zn(2)W(2)Fz, and E(2)Zn(2)W(2)Fz(2), where Fz represents the 3,4-hydrate of 5-fluorozebularine. In the absence of an inhibitor, E(2)Zn(2) is the only dimeric species detected, with no additional water molecules. Experiments conducted in H(2)(18)O indicate that the added mass W represents a trapped water molecule rather than an isobaric ammonium ion. This appears to represent the first identification of an enzyme-bound water molecule at a subunit interface (active site) using FTICR-MS. The presence of a 5-fluoro group appears to retard the decomposition of the inhibitory complex kinetically in the vapor phase, as no additional dimeric complexes (other than E(2)Zn(2)) are observed when zebularine is used in place of 5-fluorozebularine. Substrate competition assays show that in solution zebularine is released from CDA (k(off) > 0.14 s(-1)) much more rapidly than is 5-fluorozebularine (k(off) = 0.014 s(-1)), despite the greater thermodynamic stability of the zebularine complex.

Reaction of cis-3-chloroacrylic acid dehalogenase with an allene substrate, 2,3-butadienoate: hydration via an enamine.

cis-3-Chloroacrylic acid dehalogenase (cis-CaaD) catalyzes the hydrolytic dehalogenation of cis-3-haloacrylates to yield malonate semialdehyde. The enzyme processes other substrates including an allene (2,3-butadienoate) to produce acetoacetate. In the course of a stereochemical analysis of the cis-CaaD-catalyzed reaction using this allene, the enzyme was unexpectedly inactivated in the presence of NaBH(4) by the reduction of a covalent enzyme-substrate bond. Covalent modification was surprising because the accumulated evidence for cis-CaaD dehalogenation favored a mechanism involving direct substrate hydration mediated by Pro-1. However, the results of subsequent mechanistic, pre-steady state and full progress kinetic experiments are consistent with a mechanism in which an enamine forms between Pro-1 and the allene. Hydrolysis of the enamine or an imine tautomer produces acetoacetate. Reduction of the imine species is likely responsible for the observed enzyme inactivation. This is the first reported observation of a tautomerase superfamily member functioning by covalent catalysis. The results may suggest that some fraction of the cis-CaaD-catalyzed dehalogenation of cis-3-haloacrylates also proceeds by covalent catalysis.

Kinetic, crystallographic, and mechanistic characterization of TomN: elucidation of a function for a 4-oxalocrotonate tautomerase homologue in the tomaymycin biosynthetic pathway.

The biosynthesis of the C ring of the antitumor antibiotic agent, tomaymycin, is proposed to proceed through five enzyme-catalyzed steps from l-tyrosine. The genes encoding these enzymes have recently been cloned and their functions tentatively assigned, but there is limited biochemical evidence supporting the assignments of the last three steps. One enzyme, TomN, shows 58% pairwise sequence similarity with 4-oxalocrotonate tautomerase (4-OT), an enzyme found in a catabolic pathway for aromatic hydrocarbons. The TomN sequence includes three amino acids (Pro-1, Arg-11, and Arg-39) that have been identified as critical catalytic residues in 4-OT. However, the proposed substrate for TomN is very different from that processed by 4-OT. To establish the function and mechanism of TomN and its relationship with 4-OT, we conducted kinetic, mutagenic, and structural studies. The kinetic parameters for TomN, and four alanine mutants, P1A, R11A, R39A, and R61A, were determined using 2-hydroxymuconate, the substrate for 4-OT. The TomN-catalyzed reaction using this substrate compares favorably to that of 4-OT. In addition, the kinetic parameters for the P1A, R11A, and R39A mutants of TomN parallel the trends observed for the corresponding 4-OT mutants, implicating an analogous mechanism. A high-resolution crystal structure (1.4 Å) of TomN shows that the overall structure and the active site region are highly similar to those of 4-OT with a root-mean-square deviation of 0.81 Å. Moreover, key active site residues are positionally conserved. The combined results suggest that the tentative assignment for TomN and the proposed sequence of events in the biosynthetic pathway leading to the formation of the C ring of tomaymycin might not be correct. An alternative pathway that awaits biochemical confirmation is proposed.

The rate of spontaneous cleavage of the glycosidic bond of adenosine.

Previous estimates of the rate of spontaneous cleavage of the glycosidic bond of adenosine were determined by extrapolating the rates of the acid- and base-catalyzed reactions to neutral pH. Here we show that cleavage also proceeds through a pH-independent mechanism. Rate constants were determined as a function of temperature at pH 7 and a linear Arrhenius plot was constructed. Uncatalyzed cleavage occurs with a rate constant of 3.7x10(-12)s(-1) at 25 degrees C, and the rate enhancement generated by the corresponding glycoside hydrolase is approximately 5x10(12)-fold.

Pre-steady-state kinetic analysis of cis-3-chloroacrylic acid dehalogenase: analysis and implications.

Isomer-specific 3-chloroacrylic acid dehalogenases catalyze the hydrolytic dehalogenation of the cis- and trans-isomers of 3-chloroacrylate to yield malonate semialdehyde. These reactions represent key steps in the degradation of the nematocide, 1,3-dichloropropene. The kinetic mechanism of cis-3-chloroacrylic acid dehalogenase (cis-CaaD) has now been examined using stopped-flow and chemical-quench techniques. Stopped-flow analysis of the reaction, following the fluorescence of an active site tryptophan, is consistent with a minimal three-step model involving substrate binding, chemistry, and product release. Chemical-quench experiments show burst kinetics, indicating that product release is at least partially rate limiting. Global fitting of all of the kinetic results by simulation is best accommodated by a four-step mechanism. In the final kinetic model, the enzyme binds substrate with an immediate isomerization to an alternate fluorescent form and chemistry occurs, followed by the ordered release of two products, with the release of the first product as the rate-limiting step. Bromide ion is a competitive inhibitor of the reaction indicating that it binds to the free enzyme rather than to the enzyme with one product still bound. This observation suggests that malonate semialdehyde is the first product released by the enzyme (rate limiting), followed by halide. A comparison of the unliganded cis-CaaD crystal structure with that of an inactivated cis-CaaD where the prolyl nitrogen of Pro-1 is covalently attached to (R)-2-hydroxypropanoate provides a possible explanation for the isomerization step. The structure of the covalently modified enzyme shows that a seven-residue loop comprised of residues 32-38 is closed down on the active site cavity where the backbone amides of two residues (Phe-37 and Leu-38) interact with the carboxylate group of the adduct. In the unliganded form, the same loop points away from the active site cavity. Similarly, substrate binding may cause this loop to close down on the active site and sequester the reaction from the external environment.

Synthesis and conformational analysis of locked carbocyclic analogues of 1,3-diazepinone riboside, a high-affinity cytidine deaminase inhibitor.

Cytidine deaminase (CDA) catalyzes the deamination of cytidine via a hydrated transition-state intermediate that results from the nucleophilic attack of zinc-bound water at the active site. Nucleoside analogues where the leaving NH(3) group is replaced by a proton and prevent conversion of the transition state to product are very potent inhibitors of the enzyme. However, stable carbocyclic versions of these analogues are less effective as the role of the ribose in facilitating formation of hydrated species is abolished. The discovery that a 1,3-diazepinone riboside (4) operated as a tight-binding inhibitor of CDA independent of hydration provided the opportunity to study novel inhibitors built as conformationally locked, carbocyclic 1,3-diazepinone nucleosides to determine the enzyme's conformational preference for a specific form of sugar pucker. This work describes the synthesis of two target bicyclo[3.1.0]hexane nucleosides, locked as north (5) and south (6) conformers, as well as a flexible analogue (7) built with a cyclopentane ring. The seven-membered 1,3-diazepinone ring in all the three targets was built from the corresponding benzoyl-protected carbocyclic bis-allyl ureas by ring-closing metathesis. The results demonstrate CDA's binding preference for a south sugar pucker in agreement with the high-resolution crystal structures of other CDA inhibitors bound at the active site.

Contrasting behavior of conformationally locked carbocyclic nucleosides of adenosine and cytidine as substrates for deaminases.

In addition to the already known differences between adenosine deaminase (ADA) and cytidine deaminase (CDA) in terms of their tertiary structure, the sphere of Zn(+2) coordination, and their reverse stereochemical preference, we present evidence that the enzymes also differ significantly in terms of the North/South conformational preferences for their substrates and the extent to which the lack of the O(4') oxygen affects the kinetics of the enzymatic deamination of carbocyclic substrates. The carbocyclic nucleoside substrates used in this study have either a flexible cyclopentane ring or a rigid bicyclo[3.1.0]hexane scaffold.

Synthesis of conformationally locked carbocyclic 1,3-diazepinone nucleosides as inhibitors of cytidine deaminase.

We synthesized a series of carbocyclic nucleoside inhibitors of cytidine deaminase (CDA) based on a seven-membered 1,3-diazepin-2-one moiety. In the key step, the seven-membered ring was formed by a ring-closing-metathesis reaction. Therefore, the bis-allyl-urea moiety had to be protected by benzoylation in order to obtain an orientation suitable for ring closure. To our surprise, the analogue built on a flexible sugar template (4) showed a 100-fold stronger inhibition of CDA than the derivative with the preferred south-conformation.

Free-flow electrophoresis for top-down proteomics by Fourier transform ion cyclotron resonance mass spectrometry.

High-efficiency prefractionation of complex protein mixtures is critical for top-down proteomics, i.e., the analysis of intact proteins by MS. Free-flow electrophoresis (FFE) can be used for IEF to separate proteins within a pH gradient according to their pIs. In an FFE system, this separation is performed entirely in the liquid phase, without the need for particulate chromatographic media, gels, or membranes. Herein, we demonstrated the compatibility of IEF-FFE with ESI-Fourier transform ICR MS (ESI-FTICR-MS) for top-down experiments. We demonstrated that IEF-FFE of intact proteins were highly reproducible between FFE instruments, between laboratories, and between analyses. Applying native (0.2% hydroxypropylmethyl cellulose) IEF-FFE to an enzyme resulted in no decrease in enzyme activity; applying either native or denaturing (8 M urea) IEF-FFE to a four-protein mixture with different pIs resulted in isolation of each protein into separate fractions in a 96-well plate. After desalting, each protein was sequenced by top-down MS/MS. As an application of this technique, chicken erythrocyte histone H2A-IV and its major modified forms were enriched by IEF-FFE. Top-down analysis revealed Lys-5 to be a major acetylation site, in addition to N-terminal acetylation.

Rates of spontaneous disintegration of DNA and the rate enhancements produced by DNA glycosylases and deaminases.

To estimate the relative importance of alternate routes of spontaneous degradation of DNA and the rate enhancements produced by enzymes catalyzing these reactions, rate constants and thermodynamic activation parameters for the degradation of 2'-deoxynucleosides at 25 degrees C were determined by extrapolation of rates observed in the temperature range between 90 and 200 degrees C in neutral phosphate buffer. Rates of deamination of 2'-deoxycytidine, 1-methylcytosine, and cytidine were found to be identical within experimental error (t1/2 approximately 20 years, 37 degrees C). Rate constants for deamination of 2'-deoxyadenosine and 2'-deoxyguanosine, which could not be determined directly because of rapid glycoside cleavage, were estimated by assuming that methyl replacement should generate reasonable model substrates. The rates of deamination of 9-methyladenine and 9-methylguanine were found to be similar to each other (t1/2 approximately 6000 years, 37 degrees C) and approximately 10(2)-fold slower than the rates of glycoside cleavage in 2'-deoxyadenosine and 2'-deoxyguanosine. The deamination of 2'-deoxyadenosine, 2'-deoxyguanosine, and 2'-deoxycytidine led to accelerated rates of glycoside cleavage. In the exceptional case of 2'-deoxycytidine, deamination and glycoside hydrolysis proceed at very similar rates at all temperatures. Glycoside cleavage proceeds with half-times ranging from 4 years for 2'-deoxyinosine to 40 years for 2'-deoxycytidine (37 degrees C). The rate enhancements produced by DNA glycosylases, estimated by comparison with the rates of these uncatalyzed reactions, are found to be substantially smaller than those produced by deaminases and staphylococcal nuclease.

The rate enhancement produced by the ribosome: an improved model.

As a model for mechanistic comparison with peptidyl transfer within the ribosome, the reaction of aqueous glycinamide with N-formylphenylalanine trifluoroethyl ester (fPhe-TFE) represents an improvement over earlier model reactions involving Tris. The acidity of trifluoroethanol (pKa 12.4) resembles that of tRNA (12.98) more closely than do the acidities of model reactants described earlier, and the reactivity of the simple nucleophile glycinamide is free of potential complications that arise from alternative reaction pathways available to Tris. At 25 degrees C, the uncatalyzed reaction of glycinamide with fPhe-TFE proceeds with a second-order rate constant of 3 x 10(-5) M-1 s-1; DeltaH(++) = +7.8 kcal/mol; TDeltaS(++)= -15.7 kcal/mol. The ribosomal reaction of puromycin with fMet-tRNA proceeds 3 x 107-fold more rapidly, with a second-order rate constant (kcat/Km) of 1 x 10(3) M-1 s-1; DeltaH(++) = +16.0 kcal/mol; TDeltaS(++)= +2.0 kcal/mol. That rate enhancement, an order of magnitude larger than estimated earlier, is fully explained by the more favorable entropy of activation of the ribosomal reaction. Experiments involving ethylene glycol esters suggest that neighboring -OH group effects are negligible in the presence of solvent water, which itself acts as a general base catalyst. In the desolvated interior of the ribosome, the vicinal 2'-OH group of aminoacyl-tRNA probably replaces water as a general base catalyst. But the catalytic effect of the ribosome itself is overwhelmingly entropic in origin, suggesting that the ribosome achieves its effect by physical desolvation and/or juxtaposition of the reactants in a manner conducive to peptidyl transfer.

The time required for water attack at the phosphorus atom of simple phosphodiesters and of DNA.

Phosphodiester linkages, including those that join the nucleotides of DNA, are highly resistant to spontaneous hydrolysis. The rate of water attack at the phosphorus atom of phosphodiesters is known only as an upper limit, based on the hydrolysis of the dimethyl phosphate anion. That reaction was found to proceed at least 99% by C-O cleavage, at a rate suggesting an upper limit of 10(-15) s(-1) for P-O cleavage of phosphodiester anions at 25 degrees C. To evaluate the rate enhancement produced by P-O cleaving phosphodiesterases such as staphylococcal nuclease, we decided to establish the actual value of the rate constant for P-O cleavage of a simple phosphodiester anion. In dineopentyl phosphate, C-O cleavage is sterically precluded so that hydrolysis occurs only by P-O cleavage. Measurements at elevated temperatures indicate that the dineopentyl phosphate anion undergoes hydrolysis in water with a t(1/2) of 30,000,000 years at 25 degrees C, furnishing an indication of the resistance of the internucleotide linkages of DNA to water attack at phosphorus. These results imply that staphylococcal nuclease (k(cat) = 95 s(-1)) enhances the rate of phosphodiester hydrolysis by a factor of approximately 10(17). In alkaline solution, thymidylyl-3'-5'-thymidine (TpT) has been reported to decompose 10(5)-fold more rapidly than does dineopentyl phosphate. We find however that TpT and thymidine decompose at similar rates and with similar activation parameters, to a similar set of products, at pH 7 and in 1 M KOH. We infer that the decomposition of TpT is initiated by the breakdown of thymidine, not by phosphodiester hydrolysis.