Testing electrostatic complementarity in enzyme catalysis: hydrogen bonding in the ketosteroid isomerase oxyanion hole

A longstanding proposal in enzymology is that enzymes are electrostatically and geometrically complementary to the transition states of the reactions they catalyze and that this complementarity contributes to catalysis. Experimental evaluation of this contribution, however, has been difficult. We have systematically dissected the potential contribution to catalysis from electrostatic complementarity in ketosteroid isomerase. Phenolates, analogs of the transition state and reaction intermediate, bind and accept two hydrogen bonds in an active site oxyanion hole. The binding of substituted phenolates of constant molecular shape but increasing p K _a models the charge accumulation in the oxyanion hole during the enzymatic reaction. As charge localization increases, the NMR chemical shifts of protons involved in oxyanion hole hydrogen bonds increase by 0.50–0.76 ppm/p K _a unit, suggesting a bond shortening of ˜0.02 Å/p K _a unit. Nevertheless, there is little change in binding affinity across a series of substituted phenolates (ΔΔG = −0.2 kcal/mol/p K _a unit). The small effect of increased charge localization on affinity occurs despite the shortening of the hydrogen bonds and a large favorable change in binding enthalpy (ΔΔH = −2.0 kcal/mol/p K _a unit). This shallow dependence of binding affinity suggests that electrostatic complementarity in the oxyanion hole makes at most a modest contribution to catalysis of ˜300-fold. We propose that geometrical complementarity between the oxyanion hole hydrogen-bond donors and the transition state oxyanion provides a significant catalytic contribution, and suggest that KSI, like other enzymes, achieves its catalytic prowess through a combination of modest contributions from several mechanisms rather than from a single dominant contribution.     

Misunderstanding the preorganization concept can lead to confusions about the origin of enzyme catalysis

Understanding the origin of the catalytic power of enzymes has both conceptual and practical importance. One of the most important finding from computational studies of enzyme catalysis is that a major part of the catalytic power is due to the preorganization of the enzyme active site. Unfortunately, misunderstanding of the nontrivial preorganization idea lead some to assume that it does not consider the effect of the protein residues. This major confusion reflects a misunderstanding of the statement that the interaction energy of the enzyme group and the transition state (TS) is similar to the corresponding interaction between the water molecules (in the reference system) and the TS, and that the catalysis is due to the reorganization free energy of the water molecules. Obviously, this finding does not mean that we do not consider the enzyme groups. Another problem is the idea that catalysis is due to substrate preorganization. This more traditional idea is based in some cases on inconsistent interpretation of the action of model compounds, which unfortunately, do not reflect the actual situation in the enzyme active site. The present article addresses the above problems, clarifying first the enzyme polar preorganization idea and the current misunderstandings. Next we take a specific model compound that was used to promote the substrate preorganization proposal and establish its irrelevance to enzyme catalysis. Overall, we show that the origin of the catalytic power of enzymes cannot be assessed uniquely without computer simulations, since at present this is the only way of relating structure and energetics.     

Direct measurement of local and global contributions in the binding of coformycin to bovine adenosine deaminase

A general method is outlined that determines quantitatively the extent to which tight ligand binding to an enzyme active site is facilitated by the adoption of a stabler macromolecular conformation in the complex. The method therefore rejects the general assumption that competitive inhibitor binding to enzyme active sites involves only local (active site) interactions. The procedure involves comparing the unfolding transition state free energies of the free and complexed enzyme from physiological conditions. For the interaction of the transition state analog coformycin with bovine adenosine deaminase we observed that the binding free energy by the physiological enzyme was approximately 92% due to the assumption of a stabler enzyme conformation in the complex. The significance of these findings in terms of general enzyme catalysis is discussed.     

On the binding determinants of the glutamate agonist with the glutamate receptor ligand binding domain

Ionotropic glutamate receptors (GluRs) are ligand-gated membrane channel proteins found in the central neural system that mediate a fast excitatory response of neurons. In this paper, we report theoretical analysis of the ligand-protein interactions in the binding pocket of the S1S2 (ligand binding) domain of the GluR2 receptor in the closed conformation. By utilizing several theoretical methods ranging from continuum electrostatics to all-atom molecular dynamics simulations and quantum chemical calculations, we were able to characterize in detail glutamate agonist binding to the wild-type and E705D mutant proteins. A theoretical model of the protein-ligand interactions is validated via direct comparison of theoretical and Fourier transform infrared spectroscopy (FTIR) measured frequency shifts of the ligand's carboxylate group vibrations [Jayaraman et al. (2000) Biochemistry 39, 8693-8697; Cheng et al. (2002) Biochemistry 41, 1602-1608]. A detailed picture of the interactions in the binding site is inferred by analyzing contributions to vibrational frequencies produced by protein residues forming the ligand-binding pocket. The role of mobility and hydrogen-bonding network of water in the ligand-binding pocket and the contribution of protein residues exposed in the binding pocket to the binding and selectivity of the ligand are discussed. It is demonstrated that the molecular surface of the protein in the ligand-free state has mainly positive electrostatic potential attractive to the negatively charged ligand, and the potential produced by the protein in the ligand-binding pocket in the closed state is complementary to the distribution of the electrostatic potential produced by the ligand itself. Such charge complementarity ensures specificity to the unique charge distribution of the ligand.     

Calculations of antibody-antigen interactions: microscopic and semi-microscopic evaluation of the free energies of binding of phosphorylcholine analogs to McPC603.

The study of antibody-antigen interactions should greatly benefit from the development of quantitative models for the evaluation of binding free energies in proteins. The present work addresses this challenge by considering the test case of the binding free energies of phosphorylcholine analogs to the murine myeloma protein McPC603. This includes the evaluation of the differential binding energy as well as the absolute binding energies and their corresponding electrostatic contributions. Four different approaches are examined: the Protein Dipoles Langevin Dipoles (PDLD) method, the semi-microscopic PDLD (PDLD/S) method, a free energy perturbation (FEP) method based on an adiabatic charging procedure and a linear response approximation that accelerates the FEP calculation. The PDLD electrostatic calculations are augmented by estimates of the relevant hydrophobic and steric contributions. The determination of the hydrophobic energy involves an approach which considers the modification of the effective surface area of the solute by local field effects. The steric contributions are analyzed in terms of the corresponding reorganization energies. This treatment, which considers the protein as a harmonic system, views the steric forces as the restoring forces for the electrostatic interactions. The FEP method is found to give unreliable results with regular cut-off radii and starts to give quantitative results only in very expensive treatment with very large cut-off radii. The PDLD and PDLD/S methods are much faster than the FEP approach and give reasonable results for both the relative and absolute binding energies. The speed and simplicity of the PDLD/S method make it an effective strategy for interactive docking studies and indeed such an option is incorporated in the program MOLARIS. A component analysis of the different energy contributions of the FEP treatment and a similar PDLD analysis indicate that electrostatic effects provide the largest contribution to the differential binding energy, while the hydrophobic and steric contributions are much smaller. This finding lends further support to the idea that electrostatic interactions play a major role in determining the antigen specificity of McPC603.     

Comparison of calculation and experiment implicates significant electrostatic contributions to the binding stability of barnase and barstar

The contributions of electrostatic interactions to the binding stability of barnase and barstar were studied by the Poisson-Boltzmann model with three different protocols: a), the dielectric boundary specified as the van der Waals (vdW) surface of the protein along with a protein dielectric constant (epsilon (p)) of 4; b), the dielectric boundary specified as the molecular (i.e., solvent-exclusion (SE)) surface along with epsilon (p) = 4; and c), "SE + epsilon (p) = 20." The "vdW + epsilon (p) = 4" and "SE + epsilon (p) = 20" protocols predicted an overall electrostatic stabilization whereas the "SE + epsilon (p) = 4" protocol predicted an overall electrostatic destabilization. The "vdW + epsilon (p) = 4" protocol was most consistent with experiment. It quantitatively reproduced the observed effects of 17 mutations neutralizing charged residues lining the binding interface and the measured coupling energies of six charge pairs across the interface and reasonably rationalized the experimental ionic strength and pH dependences of the binding constant. In contrast, the "SE + epsilon (p) = 4" protocol predicted significantly larger coupling energies of charge pairs whereas the "SE + epsilon (p) = 20" protocol did not predict any pH dependence. This study calls for further scrutiny of the different Poisson-Boltzmann protocols and demonstrates potential danger in drawing conclusions on electrostatic contributions based on a particular calculation protocol.     

Use of intrinsic binding energy for catalysis by a cofactor-independent DNA enzyme

The concept of the Circe effect, according to which an enzyme's substrate-binding energy is utilized to destabilize the substrate towards the reaction transition state, has been shown to be a relevant catalytic strategy for naturally occurring protein enzymes and for two ribozymes that use nucleotide-based substrates and metal ion cofactors. We wished to investigate whether such a catalytic strategy extends even to divergent and unevolved catalysts constructed from biopolymers. We examined the properties of a small, in vitro selected, and cofactor-independent DNA enzyme, PS5.M, which catalyzes porphyrin metallation. The metallation reaction is unique, in that the energies for binding and for metallation of both the substrate and of a transition-state analogue (TSA) can be measured. We report that PS5.M, originally selected for binding to the TSA, displays the Circe effect in channeling a significant component of entropy-rich "intrinsic" binding energy to distort and to alter the basicity of the bound substrate. The study demonstrates that nucleic acids are, by themselves, capable of creating active sites for the catalysis of chemical reactions involving non-nucleotide substrates. Furthermore, the study of the metallation of the TSA provides a quantitative estimate of the effectiveness of such a compound in mimicking the true transition state for porphyrin metallation.     

Electrostatic contributions to residue-specific protonation equilibria and proton binding capacitance for a small protein

Charge-charge interactions in proteins are important in a host of biological processes. Here we use 13C NMR chemical shift data for individual aspartate and glutamate side chain carboxylate groups to accurately detect site-specific protonation equilibria in a variant of the B1 domain of protein G (PGB1-QDD). Carbon chemical shifts are dominated by changes in the electron distribution within the side chain and therefore excellent reporters of the charge state of individual groups, and the data are of high precision. We demonstrate that it is possible to detect local charge interactions within this small protein domain that stretch and skew the chemical shift titration curves away from "ideal" behavior and introduce a framework for the analysis of such convoluted data to study local charge-charge interactions and electrostatic coupling. It is found that, due to changes in electrostatic potential, the proton binding affinity, Ka, of each carboxyl group changes throughout the titration process and results in a linearly pH dependent pKa value. This result could be readily explained by calculations of direct charge-charge interactions based on Coulomb's law. In addition, the slope of pKa versus pH was dependent on screening by salt, and this dependence allowed the selective study of charge-charge interactions. For PGB1-QDD, it was established that mainly differences in self-energy, and not direct charge-charge interactions, are responsible for shifted pKa values within the protein environment.     

Examining methods for calculations of binding free energies: LRA, LIE, PDLD-LRA, and PDLD/S-LRA calculations of ligands binding to an HIV protease

Several strategies for evaluation of the protein-ligand binding free energies are examined. Particular emphasis is placed on the Linear Response Approximation (LRA) (Lee et. al., Prot Eng 1992;5:215-228) and the Linear Interaction Energy (LIE) method (Aqvist et. al., Prot Eng 1994;7:385-391). The performance of the Protein Dipoles Langevin Dipoles (PDLD) method and its semi-microscopic version (the PDLD/S method) is also considered. The examination is done by using these methods in the evaluating of the binding free energies of neutral C2-symmetric cyclic urea-based molecules to Human Immunodeficiency Virus (HIV) protease. Our starting point is the introduction of a thermodynamic cycle that decomposes the total binding free energy to electrostatic and non-electrostatic contributions. This cycle is closely related to the cycle introduced in our original LRA study (Lee et. al., Prot Eng 1992;5:215-228). The electrostatic contribution is evaluated within the LRA formulation by averaging the protein-ligand (and/or solvent-ligand) electrostatic energy over trajectories that are propagated on the potentials of both the polar and non-polar (where all residual charges are set to zero) states of the ligand. This average involves a scaling factor of 0.5 for the contributions from each state and this factor is being used in both the LRA and LIE methods. The difference is, however, that the LIE method neglects the contribution from trajectories over the potential of the non-polar state. This approximation is entirely valid in studies of ligands in water but not necessarily in active sites of proteins. It is found in the present case that the contribution from the non-polar states to the protein-ligand binding energy is rather small. Nevertheless, it is clearly expected that this term is not negligible in cases where the protein provides preorganized environment to stabilize the residual charges of the ligand. This contribution can be particularly important in cases of charged ligands. The analysis of the non-electrostatic term is much more complex. It is concluded that within the LRA method one has to complete the relevant thermodynamic cycle by evaluating the binding free energy of the "non-polar" ligand, l;, where all the residual charges are set to zero. It is shown that the LIE term, which involves the scaling of the van der Waals interaction by a constant beta (usually in the order of 0.15 to 0.25), corresponds to this part of the cycle. In order to elucidate the nature of this non-electrostatic term and the origin of the scaling constant beta, it is important to evaluate explicitly the different contributions to the binding energy of the non-polar ligand, DeltaG(bind,l;). Since this cannot be done at present (for relatively large ligands) by rigorous free energy perturbation approaches, we evaluate DeltaG(bind,l;) by the PDLD approach, augmented by microscopic calculations of the change in configurational entropy upon binding. This evaluation takes into account the van der Waals, hydrophobic, water penetration and entropic contributions, which are the most important free energy contributions that make up the total DeltaG(bind,l;). The sum of these contributions is scaled by a factor straight theta and it is argued that obtaining a quantitative balance between these contributions should result in straight theta = 1. By doing so we should have a reliable estimate of the value of the LIE beta and a way to understand its origin. The present approach gives straight theta values between 0.5 and 0.73, depending on the approximation used. This is encouraging but still not satisfying. Nevertheless, one might be able to use our PDLD approach to estimate the change of the LIE straight theta between different protein active sites. It is pointed out that the LIE method is quite similar to our original approach where the electrostatic term was evaluated by the LRA method and the non-electrostatic term by the PDLD method (with its vdw, solvation,     

Crystallographic and biochemical analysis of cocaine-degrading antibody 15A10

Catalytic antibody 15A10 hydrolyzes the benzoyl ester of cocaine to form the nonpsychoactive metabolites benzoic acid and ecgonine methylester. Here, we report biochemical and structural studies that characterize the catalytic mechanism. The crystal structure of the cocaine-hydrolyzing monoclonal antibody (mAb) 15A10 has been determined at 2.35 A resolution. The binding pocket is fairly shallow and mainly hydrophobic but with a cluster of three hydrogen-bond donating residues (TrpL96, AsnH33, and TyrH35). Computational docking of the transition state analogue (TSA) indicates that these residues are appropriately positioned to coordinate the phosphonate moiety of the TSA and, hence, form an oxyanion hole. Tyrosine modification of the antibody with tetranitromethane reduced hydrolytic activity to background level. The contribution from these and other residues to catalysis and TSA binding was explored by site-directed mutagenesis of 15A10 expressed in a single chain fragment variable (scFv) format. The TyrH35Phe mutant had 4-fold reduced activity, and TrpL96Ala, TrpL96His, and AsnH33Ala mutants were all inactive. Comparison with an esterolytic antibody D2.3 revealed a similar arrangement of tryptophan, asparagine, and tyrosine residues in the oxyanion hole that stabilizes the transition state for ester hydrolysis. Furthermore, the crystal structure of the bacterial cocaine esterase (cocE) also showed that the cocE employs a tyrosine hydroxyl in the oxyanion hole. Thus, the biochemical and structural data are consistent with the catalytic antibody providing oxyanion stabilization as its major contribution to catalysis.     

Electrostatic contributions to the binding free energy of the lambdacI repressor to DNA.

A model based on the nonlinear Poisson-Boltzmann (NLPB) equation is used to study the electrostatic contribution to the binding free energy of the lambdacI repressor to its operator DNA. In particular, we use the Poisson-Boltzmann model to calculate the pKa shift of individual ionizable amino acids upon binding. We find that three residues on each monomer, Glu34, Glu83, and the amino terminus, have significant changes in their pKa and titrate between pH 4 and 9. This information is then used to calculate the pH dependence of the binding free energy. We find that the calculated pH dependence of binding accurately reproduces the available experimental data over a range of physiological pH values. The NLPB equation is then used to develop an overall picture of the electrostatics of the lambdacI repressor-operator interaction. We find that long-range Coulombic forces associated with the highly charged nucleic acid provide a strong driving force for the interaction of the protein with the DNA. These favorable electrostatic interactions are opposed, however, by unfavorable changes in the solvation of both the protein and the DNA upon binding. Specifically, the formation of a protein-DNA complex removes both charged and polar groups at the binding interface from solvent while it displaces salt from around the nucleic acid. As a result, the electrostatic contribution to the lambdacI repressor-operator interaction opposes binding by approximately 73 kcal/mol at physiological salt concentrations and neutral pH. A variety of entropic terms also oppose binding. The major force driving the binding process appears to be release of interfacial water from the protein and DNA surfaces upon complexation and, possibly, enhanced packing interactions between the protein and DNA in the interface. When the various nonelectrostatic terms are described with simple models that have been applied previously to other binding processes, a general picture of protein/DNA association emerges in which binding is driven by the nonpolar interactions, whereas specificity results from electrostatic interactions that weaken binding but are necessary components of any protein/DNA complex.     

Multiple drugs and multiple targets: An analysis of the electrostatic determinants of binding between non‐nucleoside HIV‐1 reverse transcriptase inhibitors and variants of HIV‐1 RT

We present a systematic, computational analysis of the electrostatic component of binding of three HIV‐1 RT inhibitors—nevirapine (NVP), efavirenz (EFV), and the recently approved rilpivirine (RPV)—to wild‐type (WT) and mutant variants of RT. Electrostatic charge optimization was applied to determine how suited each molecule's charge distribution is for binding WT and individual mutants of HIV‐1 RT. Although the charge distributions of NVP and EFV are rather far from being optimal for tight binding, RPVs charge distribution is close to the theoretical, optimal charge distribution for binding WT HIV‐1 RT, although slight changes in charge can dramatically impact binding energetics. Moreover, toward the L100I/K103N double mutant, RPVs charge distribution is quite far from optimal. We also determine the contributions of chemical moieties on each molecule toward the electrostatic component of binding and show that different regions of a drug molecule may be used for recognition by different RT variants. The electrostatic contributions of certain RT residues toward drug binding are also computed to highlight critical residues for each interaction. Finally, the charge distribution of RPV is optimized to promiscuously bind to three RT variants rather than to each one in turn, with the resulting charge distribution being a compromise between the optimal charge distributions to each individual variant. Taken together, this work demonstrates that even in a binding site considered quite hydrophobic, electrostatics play a subtle yet varying role that must be considered in designing next‐generation molecules that recognize rapidly mutating targets. Proteins 2012. © 2011 Wiley Periodicals, Inc.     

Separation of factors responsible for lysozyme catalysis

The four major factors contributing to lysozyme facilitation of acetal hydrolysis are general acid catalysis, distortion, electrostatic stabilization of the transition state and entropy effects. The importance of general acid catalysis is assessed from the Brønsted relationship using an a coefficient of 0·5 based on model compounds. The degree of electrostatic stabilization is assessed from the electric field interactions of Asp 52 and Glu 35. Interaction of lysozyme with transition state analogs is used to calculate the contribution of distortion to catalysis. The remaining acceleration factor is assigned to entropy effects. The value of the entropy effect suggests that the transition state has some freedom of motion on the enzyme.     

On the relationship between thermal stability and catalytic power of enzymes

The possible relationship between the thermal stability and the catalytic power of enzymes is of great current interest. In particular, it has been suggested that thermophilic or hyperthermophilic (Tm) enzymes have lower catalytic power at a given temperature than the corresponding mesophilic (Ms) enzymes, because the thermophilic enzymes are less flexible (assuming that flexibility and catalysis are directly correlated). These suggestions presume that the reduced dynamics of the thermophilic enzymes is the reason for their reduced catalytic power. The present paper takes the specific case of dihydrofolate reductase (DHFR) and explores the validity of the above argument by simulation approaches. It is found that the Tm enzymes have restricted motions in the direction of the folding coordinate, but this is not relevant to the chemical process, since the motions along the reaction coordinate are perpendicular to the folding motions. Moreover, it is shown that the rate of the chemical reaction is determined by the activation barrier and the corresponding reorganization energy, rather than by dynamics or flexibility in the ground state. In fact, as far as flexibility is concerned, we conclude that the displacement along the reaction coordinate is larger in the Tm enzyme than in the Ms enzyme and that the general trend in enzyme catalysis is that the best catalyst involves less motion during the reaction than the less optimal catalyst. The relationship between thermal stability and catalysis appears to reflect the fact that to obtain small electrostatic reorganization energy it is necessary to invest some folding energy in the overall preorganization process. Thus, the optimized catalysts are less stable. This trend is clearly observed in the DHFR case.     

Prechemistry versus preorganization in DNA replication fidelity

The molecular origin of nucleotide insertion catalysis and fidelity of DNA polymerases is explored by means of computational simulations. Special attention is paid to the examination of the validity of proposals that invoke prechemistry effects, checkpoints concepts, and dynamical effects. The simulations reproduce the observed fidelity in Pol β, starting with the relevant observed X-ray structures of the complex with the right (R) and wrong (W) nucleotides. The generation of free energy surfaces for the R and W systems also allowed us to analyze different proposals about the origin of the fidelity and to reach several important conclusions. It is found that the potential of mean force (PMF) obtained by proper sampling does not support QM/MM-based proposals of a large barrier before the prechemistry state. Furthermore, examination of dynamical proposals by the renormalization approach indicates that the motions from open to close configurations do not contribute to catalysis or fidelity. Finally we discuss and analyze the induced fit concept and show that, despite its importance, it does not explain fidelity. That is, the fidelity is apparently due to the change in the preorganization of the chemical site, as a result of the relaxation of the binding site upon binding of the incorrect nucleotide. Finally and importantly, since the issue is the barrier associated with the enzyme-substrate (ES)/DNA complex at the chemical transition state and not the path to this complex formation (unless this path involves rate determining steps), it is also not useful to invoke checkpoints while discussing fidelity.     

Ground State Destabilization by Anionic Nucleophiles Contributes to the Activity of Phosphoryl Transfer Enzymes

Enzymes stabilize transition states of reactions while limiting binding to ground states, as is generally required for any catalyst. Alkaline Phosphatase (AP) and other nonspecific phosphatases are some of Nature''s most impressive catalysts, achieving preferential transition state over ground state stabilization of more than 10²²-fold while utilizing interactions with only the five atoms attached to the transferred phosphorus. We tested a model that AP achieves a portion of this preference by destabilizing ground state binding via charge repulsion between the anionic active site nucleophile, Ser102, and the negatively charged phosphate monoester substrate. Removal of the Ser102 alkoxide by mutation to glycine or alanine increases the observed P_i affinity by orders of magnitude at pH 8.0. To allow precise and quantitative comparisons, the ionic form of bound P_i was determined from pH dependencies of the binding of P_i and tungstate, a P_i analog lacking titratable protons over the pH range of 5–11, and from the ³¹P chemical shift of bound P_i. The results show that the P_i trianion binds with an exceptionally strong femtomolar affinity in the absence of Ser102, show that its binding is destabilized by ≥10⁸-fold by the Ser102 alkoxide, and provide direct evidence for ground state destabilization. Comparisons of X-ray crystal structures of AP with and without Ser102 reveal the same active site and P_i binding geometry upon removal of Ser102, suggesting that the destabilization does not result from a major structural rearrangement upon mutation of Ser102. Analogous P_i binding measurements with a protein tyrosine phosphatase suggest the generality of this ground state destabilization mechanism. Our results have uncovered an important contribution of anionic nucleophiles to phosphoryl transfer catalysis via ground state electrostatic destabilization and an enormous capacity of the AP active site for specific and strong recognition of the phosphoryl group in the transition state.     

Role of an active site guanine in hairpin ribozyme catalysis probed by exogenous nucleobase rescue

The hairpin ribozyme is a small catalytic RNA with reversible phosphodiester cleavage activity. Biochemical and structural studies exclude a requirement for divalent metal cation cofactors and implicate one active site nucleobase in particular, G8, in the catalytic mechanism. Our previous work demonstrated that the cleavage activity that is lost when G8 is replaced by an abasic residue is restored when certain nucleobases are provided in solution. The specificity and pH dependence of exogenous nucleobase rescue were consistent with several models of the rescue mechanism, including general acid base catalysis, electrostatic stabilization of negative charge in the transition state or a requirement for protonation to facilitate exogenous nucleobase binding. Detailed analyses of exogenous nucleobase rescue for both cleavage and ligation reactions now allow us to refine models of the rescue mechanism. Activity increased with increasing pH for both unmodified ribozyme reactions and unrescued reactions of abasic variants lacking G8. This similarity in pH dependence argues against a role for G8 as a general base catalyst, because G8 deprotonation could not be responsible for the pH-dependent transition in the abasic variant. Exogenous nucleobase rescue of both cleavage and ligation activity increased with decreasing pH, arguing against a role for rescuing nucleobases in general acid catalysis, because a nucleobase that contributes general acid catalysis in the cleavage pathway should provide general base catalysis in ligation. Analysis of the concentration dependence of cytosine rescue at high and low pH demonstrated that protonation promotes catalysis within the nucleobase-bound ribozyme complex but does not stabilize nucleobase binding in the ground state. These results support an electrostatic stabilization mechanism in which exogenous nucleobase binding counters negative charge that develops in the transition state.     

Entropy-driven binding of picomolar transition state analogue inhibitors to human 5'-methylthioadenosine phosphorylase

Human 5'-methylthioadenosine phosphorylase (MTAP) links the polyamine biosynthetic and S-adenosyl-l-methionine salvage pathways and is a target for anticancer drugs. p-Cl-PhT-DADMe-ImmA is a 10 pM, slow-onset tight-binding transition state analogue inhibitor of the enzyme. Titration of homotrimeric MTAP with this inhibitor established equivalent binding and independent catalytic function of the three catalytic sites. Thermodynamic analysis of MTAP with tight-binding inhibitors revealed entropic-driven interactions with small enthalpic penalties. A large negative heat capacity change of -600 cal/(mol K) upon inhibitor binding to MTAP is consistent with altered hydrophobic interactions and release of water. Crystal structures of apo MTAP and MTAP in complex with p-Cl-PhT-DADMe-ImmA were determined at 1.9 and 2.0 ? resolution, respectively. Inhibitor binding caused condensation of the enzyme active site, reorganization at the trimer interfaces, the release of water from the active sites and subunit interfaces, and compaction of the trimeric structure. These structural changes cause the entropy-favored binding of transition state analogues. Homotrimeric human MTAP is contrasted to the structurally related homotrimeric human purine nucleoside phosphorylase. p-Cl-PhT-DADMe-ImmA binding to MTAP involves a favorable entropy term of -17.6 kcal/mol with unfavorable enthalpy of 2.6 kcal/mol. In contrast, binding of an 8.5 pM transition state analogue to human PNP has been shown to exhibit the opposite behavior, with an unfavorable entropy term of 3.5 kcal/mol and a favorable enthalpy of -18.6 kcal/mol. Transition state analogue interactions reflect protein architecture near the transition state, and the profound thermodynamic differences for MTAP and PNP suggest dramatic differences in contributions to catalysis from protein architecture.     

Continuum electrostatic model for the binding of cytochrome c2 to the photosynthetic reaction center from Rhodobacter sphaeroides

Electrostatic interactions are important for protein-protein association. In this study, we examined the electrostatic interactions between two proteins, cytochrome c(2) (cyt c(2)) and the reaction center (RC) from the photosynthetic bacterium Rhodobacter sphaeroides, that function in intermolecular electron transfer in photosynthesis. Electrostatic contributions to the binding energy for the cyt c(2)-RC complex were calculated using continuum electrostatic methods based on the recent cocrystal structure [Axelrod, H. L., et al. (2002) J. Mol. Biol. 319, 501-515]. Calculated changes in binding energy due to mutations of charged interface residues agreed with experimental results for a protein dielectric constant epsilon(in) of 10. However, the electrostatic contribution to the binding energy for the complex was close to zero due to unfavorable desolvation energies that compensate for the favorable Coulomb attraction. The electrostatic energy calculated as a function of displacement of the cyt c(2) from the bound position showed a shallow minimum at a position near but displaced from the cocrystal configuration. These results show that although electrostatic steering is present, other short-range interactions must be present to contribute to the binding energy and to determine the structure of the complex. Calculations made to model the experimental data on association rates indicate a solvent-separated transition state for binding in which the cyt c(2) is displaced approximately 8 A above its position in the bound complex. These results are consistent with a two-step model for protein association: electrostatic docking of the cyt c(2) followed by desolvation to form short-range van der Waals contacts for rapid electron transfer.