Energetics of ribonuclease A catalysis. 1. pH, ionic strength, and solvent isotope dependence of the hydrolysis of cytidine cyclic 2',3'-phosphate

The pH, ionic strength, and solvent deuterium isotope dependence of the steady-state kinetics of the ribonuclease A catalyzed hydrolysis of cytidine cyclic 2',3'-phosphate has been investigated by using, primarily, the technique of flow microcalorimetry to monitor the kinetics. The pH dependence of the Michaelis-Menten parameters has been analyzed by assuming the participation of His-12 and -119 of the enzyme and a third ionizing group, postulated to be on the pyrimidine ring of the substrate, to determine the pH-independent rate constant kc, and Michaelis constant Km. The reported pH analysis, together with existing NMR data and chemical modification studies, allows an assignment of the functional roles of His-12 and -119 as being those of general acid and general base catalytic residues, respectively. At high pH, the apparent Km value is found to increase to unity. This drop in affinity between the enzyme and the substrate at high pH indicates that the substrate binds to the enzyme primarily through an electrostatic interaction with the active-site histidine residues, particularly His-12. The apparent absence of an interaction with the riboside portion of the substrate is suggested to be due to the fact that the substrate exists in a syn conformation about its glycosidic bond and thus cannot interact optimally with the enzyme's binding pocket. This will result in a relative destabilization of the enzyme-substrate complex, which can then be relieved upon the formation of the transition state. The ionic strength dependence of ribonuclease activity is shown to be primarily a result of its effect on the pKa of the histidine residues and a concomitant change in the value of Km.     

Temperature effects on the catalytic efficiency, rate enhancement, and transition state affinity of cytidine deaminase, and the thermodynamic consequences for catalysis of removing a substrate "anchor"

To obtain a clearer understanding of the forces involved in transition state stabilization by Escherichia coli cytidine deaminase, we investigated the thermodynamic changes that accompany substrate binding in the ground state and transition state for substrate hydrolysis. Viscosity studies indicate that the action of cytidine deaminase is not diffusion-limited. Thus, K(m) appears to be a true dissociation constant, and k(cat) describes the chemical reaction of the ES complex, not product release. Enzyme-substrate association is accompanied by a loss of entropy and a somewhat greater release of enthalpy. As the ES complex proceeds to the transition state (ES), there is little further change in entropy, but heat is taken up that almost matches the heat that was released with ES formation. As a result, k(cat)/K(m) (describing the overall conversion of the free substrate to ES is almost invariant with changing temperature. The free energy barrier for the enzyme-catalyzed reaction (k(cat)/K(m)) is much lower than that for the spontaneous reaction (k(non)) (DeltaDeltaG = -21.8 kcal/mol at 25 degrees C). This difference, which also describes the virtual binding affinity of the enzyme for the activated substrate in the transition state (S), is almost entirely enthalpic in origin (DeltaDeltaH = -20.2 kcal/mol), compatible with the formation of hydrogen bonds that stabilize the ES complex. Thus, the transition state affinity of cytidine deaminase increases rapidly with decreasing temperature. When a hydrogen bond between Glu-91 and the 3'-hydroxyl moiety of cytidine is disrupted by truncation of either group, k(cat)/K(m) and transition state affinity are each reduced by a factor of 10(4). This effect of mutation is entirely enthalpic in origin (DeltaDeltaH approximately 7.9 kcal/mol), somewhat offset by a favorable change in the entropy of transition state binding. This increase in entropy is attributed to a loss of constraints on the relative motions of the activated substrate within the ES complex. In an Appendix, some objections to the conventional scheme for transition state binding are discussed.     

Site-bound water and the shortcomings of a less than perfect transition state analogue

Kinetic measurements have shown that substantial enthalpy changes accompany substrate binding by cytidine deaminase, increasing markedly as the reaction proceeds from the ground state (1/K(m), DeltaH = -13 kcal/mol) to the transition state (1/K(tx), DeltaH = -20 kcal/mol) [Snider, M. J., et al. (2000) Biochemistry 39, 9746-9753]. In the present work, we determined the thermodynamic changes associated with the equilibrium binding of inhibitors by cytidine deaminase by isothermal titration calorimetry and van't Hoff analysis of the temperature dependence of their inhibition constants. The results indicate that the binding of the transition state analogue 3,4-dihydrouridine DeltaH = -21 kcal/mol), like that of the transition state itself (DeltaH = -20 kcal/mol), is associated with a large favorable change in enthalpy. The significantly smaller enthalpy change that accompanies the binding of 3,4-dihydrozebularine (DeltaH = -10 kcal/mol), an analogue of 3,4-dihydrouridine in which a hydrogen atom replaces this inhibitor's 4-OH group, is consistent with the view that polar interactions with the substrate at the site of its chemical transformation play a critical role in reducing the enthalpy of activation for substrate hydrolysis. The entropic shortcomings of 3,4-dihydrouridine, in capturing all of the free energy involved in binding the actual transition state, may arise from its inability to displace a water molecule that occupies the binding site normally occupied by product ammonia.     

Energetics of ribonuclease A catalysis. 2. Nonenzymatic hydrolysis of cytidine cyclic 2',3'-phosphate

Various kinetic aspects of the nonenzymatic hydrolysis of cytidine cyclic 2',3'-phosphate and uridine cyclic 2',3'-phosphate have been studied in order to provide a basis for comparison with the ribonuclease A catalyzed hydrolysis reaction. Studies of the pH dependence of the nonenzymatic reaction reveal mechanisms that are first order in hydroxide concentration and second order in hydrogen ion concentration, in addition to a "water" reaction. The rate constant for the water reaction was found to be very small, approximately equal to 2.5 X 10(-6) min-1. General base catalyzed hydrolysis reactions were also studied with imidazole as the catalyst. At pH values in which both the protonated and neutral forms of imidazole are present, a kinetic mechanism was observed that appears to be second order in total imidazole concentration, thus suggesting that bifunctional catalysis occurs. The activation enthalpy for the hydroxide, hydrogen ion, water, and imidazole catalyzed reactions was determined.     

The interaction of purified acetylcholinesterase from pig brain with liposomes.

The binding of pig brain acetylcholinesterase to artificial phospholipid membranes was investigated at different temperatures. Calculation of the thermodynamic parameters revealed a small negative enthalpy change, but a large negative change in the free energy and a large positive change in the entropy on binding. The large entropy change might be interpreted as being responsible for forming the enzyme-membrane complex and was indicative of hydrophobic interactions between lipid and protein. This conclusion would also favour the hypothesis that the enzyme was an integral protein. Further support for this theory was provided by the study of acetylcholinesterase binding to liposomes containing the phospholipid 1,2-dimyristoyl-sn-glycero-3-phosphocholine. Lowering the temperature below the transition temperature or incorporating cholesterol into the liposomes decreased enzyme binding. Both factors could be interpreted as decreasing the fluidity of the hydrocarbon side chains of the phospholipids, causing an increase in bilayer thickness due to closer packing of side chains. This membrane condensation would certainly not favour the binding of integral protein molecules.     

Structural, thermodynamic, and kinetic analyses of tetrahydrooxazine-derived inhibitors bound to beta-glucosidases

The understanding of transition state mimicry in glycoside hydrolysis is increasingly important both in the quest for novel specific therapeutic agents and for the deduction of enzyme function and mechanism. To aid comprehension, inhibitors can be characterized through kinetic, thermodynamic, and structural dissection to build an "inhibition profile." Here we dissect the binding of a tetrahydrooxazine inhibitor and its derivatives, which display Ki values around 500 nm. X-ray structures with both a beta-glucosidase, at 2 A resolution, and an endoglucanase at atomic (approximately 1 A) resolution reveal similar interactions between the tetrahydrooxazine inhibitor and both enzymes. Kinetic analyses reveal the pH dependence of kcat/Km and 1/Ki with both enzyme systems, and isothermal titration calorimetry unveils the enthalpic and entropic contributions to beta-glucosidase inhibition. The pH dependence of enzyme activity mirrored that of 1/Ki in both enzymes, unlike the cases of isofagomine and 1-deoxynojirimycin that have been characterized previously. Calorimetric dissection reveals a large favorable enthalpy that is partially offset by an unfavorable entropy upon binding. In terms of the similar profile for the pH dependence of 1/Ki and the pH dependence of kcat/Km, the significant enthalpy of binding when compared with other glycosidase inhibitors, and the tight binding at the optimal pH of the enzymes tested, tetrahydrooxazine and its derivatives are a significantly better class of glycosidase inhibitor than previously assumed.     

The pH dependence of the thermodynamics of the interaction of 3'-cytidine monophosphate with ribonuclease A.

The apparent free energy (deltaGapp) and enthalpy changes (deltaHB) associated with the interaction of 3'-cytosine monophosphate (3'-CMP) and ribonuclease A (RNase) are reported for the pH range 4--9, T = 25 degrees, mu = 0.05. The pH dependence of deltaGapp and deltaHB has been interpreted in terms of coupled ionization of histidine residues 12, 48, and 119, assuming that only the dianionic form of the inhibitor is bound. The results of this analysis are consistent with the calorimetric and potentiometric titration results for the free enzyme and its 3'-CMP complex reported in the previous paper (M. Flogel and R. L. Biltonen ((1975), Biochemistry, preceding paper in this issue). This analysis allows the calculation of the thermodynamic quantities associated with hypothetical but clearly defined reactions (e.g., the reaction of the dianionic inhibitor with the completely protonated enzyme). It is concluded that the primary thermodynamic driving forces for the reaction are van der Waals interactions between the riboside moiety and the protein fabric and electrostatic interaction between the negatively charged phosphate group of the inhibitor and the positively charged histidine residues at the binding locus. It is also suggested that the binding reaction is weakly coupled (approximately to 0.5 kcal/mol) with a conformational change of the protein associated with protonation of residue 48. These results are consistent with the model originally proposed by G. G. Hammes ((1968), Adv. Protein Chem. 23, 1) and lend additional quantitative detail to the nature of the reaction.     

Thermodynamics of oligosaccharide binding to a monoclonal antibody specific for a Salmonella O-antigen point to hydrophobic interactions in the binding site.

The thermodynamic characteristics of oligosaccharide binding to an antibody binding site that is dominated by aromatic amino acids suggest that the hydrophobic effect contributes substantially to complex formation as well as hydrogen bonding and van der Waals interactions. A detailed titration microcalorimetric study on the temperature dependence of the binding of a trisaccharide, representing the epitope of a Salmonella O-antigen, showed that its maximum binding to the monoclonal antibody Se155-4 occurs just below room temperature and both enthalpy and entropy changes are strongly dependent on temperature in a mutually compensating manner. The heat capacity change also shows an unusually strong temperature dependence being large and negative above room temperature and positive below. van't Hoff analysis of the temperature dependence of the binding constant yielded a biphasic curve with two apparent intrinsic enthalpy estimations (approximately -100 kJ mol-1 above 18 degrees C and approximately +100 kJ mol-1 below), each very different from the calorimetrically determined enthalpies (ranging from about -60 kJ mol-1 to -20 kJ mol-1). This was interpreted as being due to large enthalpy contributions from concomitant reactions, most notably changes in solvation. Linear plots, -delta H0 versus -T delta S0, observed for temperature-dependent measurements mirror the behavior seen for a series of functional group replacements, suggesting that the molecular and physical origin of these phenomena are closely related and linked to the role of water in complex formation. The thermodynamic results are compared to the mode of binding determined from a 2.05-A resolution structure of the Fab-oligosaccharide complex, and with literature data for the heat capacities of sugars in aqueous solution and for the thermodynamics of carbohydrate binding to transport proteins and lectins.     

Structural energetics of barstar studied by differential scanning microcalorimetry.

The energetics of barstar denaturation have been studied by CD and scanning microcalorimetry in an extended range of pH and salt concentration. It was shown that, upon increasing temperature, barstar undergoes a transition to the denatured state that is well approximated by a two-state transition in solutions of high ionic strength. This transition is accompanied by significant heat absorption and an increase in heat capacity. The denaturational heat capacity increment at approximately 75 degrees C was found to be 5.6 +/- 0.3 kJ K-1 mol-1. In all cases, the value of the measured enthalpy of denaturation was notably lower than those observed for other small globular proteins. In order to explain this observation, the relative contributions of hydration and the disruption of internal interactions to the total enthalpy and entropy of unfolding were calculated. The enthalpy and entropy of hydration were found to be in good agreement with those calculated for other proteins, but the enthalpy and entropy of breaking internal interactions were found to be among the lowest for all globular proteins that have been studied. Additionally, the partial specific heat capacity of barstar in the native state was found to be 0.37 +/- 0.03 cal K-1 g-1, which is higher than what is observed for most globular proteins and suggests significant flexibility in the native state. It is known from structural data that barstar undergoes a conformational change upon binding to its natural substrate barnase.(ABSTRACT TRUNCATED AT 250 WORDS)     

The magnitude of electrostatic interactions in inhibitor binding and during catalysis by ribonuclease A.

It is demonstrated that a model of nucleotide binding to ribonuclease A similar to that proposed by Hammes and coworkers (G. G. Hammes (1968), Adv. Protein Chem. 23, 1) is at least, approximately applicable for both cyclic nucleotide substrates and mononucleotide inhibitors at pH values less than or equal to 6.5 and as a function of ionic strength. Calorimetric data on various inhibitors show that the binding reaction can be thermodynamically dissected into a contribution arising from van der Waal's interaction of the nucleoside moiety, characterized by a large negative enthalpy change, and a contribution arising from electrostatic interactions between the negatively charged phosphate group of the inhibitor and the positively charged protein fabric, characterized by a large positive unitary entropy change. Assuming a catalytic mechanism involving the formation of a dianionic pentacoordinated phosphate transition state intermediate, the magnitude of the effect of electrostatic interactions on the overall rate enhancement by the enzyme is estimated to be 2 times 10(2) to 10(6). It is suggested that this effect, along with substrate approximation effects, is sufficient to "explain" the catalytic behavior of the enzyme.     

Thermodynamic profiles of penicillin G hydrolysis catalyzed by wild-type and Met----Ala168 mutant penicillin acylases from Kluyvera citrophila

The Met-168 residue in penicillin acylase from Kluyvera citrophila was changed to Ala by oligonucleotide site-directed mutagenesis. The Ala-168 mutant exhibited different substrate specificity than wild-type and enhanced thermal stability. The thermodynamic profiles for penicillin G hydrolysis catalyzed by both enzymes were obtained from the temperature dependence of the steady-state kinetic parameters Km and kcat. The high values of enthalpy and entropy of activation determined for the binding of substrate suggest that an induced-fit-like mechanism takes place. The Met----Ala168 mutation unstabilizes the first transition-state (E..S not equal to) and the enzyme-substrate complex (ES) causing a decrease in association equilibrium and specificity constants in the enzyme. However, no change is observed in the acyl-enzyme formation. It is concluded that residue 168 is involved in the enzyme conformational rearrangements caused by the interaction of the acid moiety of the substrate at the active site.     

Temperature dependence of histidine ionization constants in myoglobin

The standard enthalpy of ionization of six titratable histidines in horse metaquomyoglobin was determined by repeating proton NMR titrations as a function of temperature and using the van't Hoff relationship. It was found that deltaH degrees varies between 16 and 37 kJ mol(-1) in the protein, compared with a value of 29 kJ mol(-1) in free histidine. The standard entropy change was evaluated by combining the enthalpy and free energy changes derived from the pKa values. Although the entropy change could not be precisely and accurately obtained by this method, it could be established that it spans a wide range, from -60 to 0 J K(-1) mol(-1), about the value of -23 J K(-1) mol(-1) for the free histidine. The entropy change was used within the framework of enthalpy-entropy compensation to partition the solvation component from the standard thermodynamic quantities for each of the titrating residues. It was shown that the partitioning of the values in the protein is not readily understood in terms of solvent accessibility or electrostatic interactions. The contribution of solvation effects to the temperature response appeared to be significant only in the case of His-119 and His-48. The standard quantities were also used to explore the energetics of proton binding in the native state at temperatures below the onset of thermal denaturation.     

Isotope effect studies of the pyruvate-dependent histidine decarboxylase from Lactobacillus 30a

The decarboxylation of histidine by the pyruvate-dependent histidine decarboxylase of Lactobacillus 30a shows a carbon isotope effect of k12/k13 = 1.0334 +/- 0.0005 and a nitrogen isotope effect k14/k15 = 0.9799 +/- 0.0006 at pH 4.8, 37 degrees C. The carbon isotope effect is slightly increased by deuteriation of the substrate and slightly decreased in D2O. The observed nitrogen isotope effect indicates that the imine nitrogen in the substrate-Schiff base intermediate complex is ordinarily protonated, and the pH dependence of the carbon isotope effect indicates that both protonated and unprotonated forms of this intermediate are capable of undergoing decarboxylation. As with the pyridoxal 5'-phosphate dependent enzyme, Schiff base formation and decarboxylation are jointly rate-limiting, with the intermediate histidine-pyruvate Schiff base showing a decarboxylation/Schiff base hydrolysis ratio of 0.5-1.0 at pH 4.8. The decarboxylation transition state is more reactant-like for the pyruvate-dependent enzyme than for the pyridoxal 5'-phosphate dependent enzyme. These studies find no particular energetic or catalytic advantage to the use of pyridoxal 5'-phosphate over covalently bound pyruvate in catalysis of the decarboxylation of histidine.     

Fluorescence quenching studies on binding fluoreno-9-spiro-oxazolidinedione to human serum albumin.

Human serum albumin fluorescence quenching by fluorene-9-spiro-oxazolidinedione has been analyzed as a function of temperature. Such temperature dependence suggests that the mechanism of the quenching process is static in origin. This type of quenching implies that a non-fluorescent complex between oxazolidinedione and serum albumin has been formed and following the Stern-Volmer relationship we have calculated the binding constant. Thermodynamic parameters were also determined. The positive and large values of entropy and the negative value for enthalpy suggest that both hydrophobic and electrostatic interactions may play an important role in the stabilization of the complex. Finally, the irreversible changes in the spectral properties of HSA are interpreted in binding terms.     

Temperature dependence of the preferential interactions of ribonuclease A in aqueous co-solvent systems: thermodynamic analysis.

The temperature dependence of preferential solvent interactions with ribonuclease A in aqueous solutions of 30% sorbitol, 0.6 M MgCl2, and 0.6 M MgSO4 at low pH (1.5 and 2.0) and high pH (5.5) has been investigated. This protein was stabilized by all three co-solvents, more so at low pH than high pH (expect 0.6 M MgCl2 at pH 5.5). The preferential hydration of protein in all three co-solvents was high at temperatures below 30 degrees C and decreased with a further increase in temperature (for 0.6 M MgCl2 at pH 5.5, this was not significant), indicating a greater thermodynamic instability at low temperature than at high temperature. The preferential hydration of denatured protein (low pH, high temperature) was always greater than that of native protein (high pH, high temperature). In 30% sorbitol, the interaction passed to preferential binding at 45% for native ribonuclease A and at 55 degrees C for the denatured protein. Availability of the temperature dependence of the variation with sorbitol concentration of the chemical potential of the protein, (delta mu(2)/delta m3)T,p,m2, permitted calculation of the corresponding enthalpy and entropy parameters. Combination with available data on sorbitol concentration dependence of this interaction parameter gave (approximate) values of the transfer enthalpy, delta H2,tr, and transfer entropy delta S2,tr. Transfer of ribonuclease A from water into 30% sorbitol is characterized by positive values of the transfer free energy, transfer enthalpy, transfer entropy, and transfer heat capacity. On denaturation, the transfer enthalpy becomes more positive. This increment, however, is small relative to both the enthalpy of unfolding in water and to the transfer enthalpy of the native protein from water a 30% sorbitol solution.     

Isothermal titration calorimetric studies on the interaction of the major bovine seminal plasma protein, PDC-109 with phospholipid membranes

The interaction of the major bovine seminal plasma protein, PDC-109 with lipid membranes was investigated by isothermal titration calorimetry. Binding of the protein to model membranes made up of diacyl phospholipids was found to be endothermic, with positive values of binding enthalpy and entropy, and could be analyzed in terms of a single type of binding sites on the protein. Enthalpies and entropies for binding to diacylphosphatidylcholine membranes increased with increase in temperature, although a clear-cut linear dependence was not observed. The entropically driven binding process indicates that hydrophobic interactions play a major role in the overall binding process. Binding of PDC-109 with dimyristoylphosphatidylcholine membranes containing 25 mol% cholesterol showed an initial increase in the association constant as well as enthalpy and entropy of binding with increase in temperature, whereas the values decreased with further increase in temperature. The affinity of PDC-109 for phosphatidylcholine increased at higher pH, which is physiologically relevant in view of the basic nature of the seminal plasma. Binding of PDC-109 to Lyso-PC could be best analysed in terms of two types of binding interactions, a high affinity interaction with Lyso-PC micelles and a low-affinity interaction with the monomeric lipid. Enthalpy-entropy compensation was observed for the interaction of PDC-109 with phospholipid membranes, suggesting that water structure plays an important role in the binding process.     

Thermodynamics of the temperature-induced unfolding of globular proteins.

The heat capacity, enthalpy, entropy, and Gibbs energy changes for the temperature-induced unfolding of 11 globular proteins of known three-dimensional structure have been obtained by microcalorimetric measurements. Their experimental values are compared to those we calculate from the change in solvent-accessible surface area between the native proteins and the extended polypeptide chain. We use proportionality coefficients for the transfer (hydration) of aliphatic, aromatic, and polar groups from gas phase to aqueous solution, we estimate vibrational effects, and we discuss the temperature dependence of each constituent of the thermodynamic functions. At 25 degrees C, stabilization of the native state of a globular protein is largely due to two favorable terms: the entropy of non-polar group hydration and the enthalpy of interactions within the protein. They compensate the unfavorable entropy change associated with these interactions (conformational entropy) and with vibrational effects. Due to the large heat capacity of nonpolar group hydration, its stabilizing contribution decreases quickly at higher temperatures, and the two unfavorable entropy terms take over, leading to temperature-induced unfolding.     

Interaction of membrane phospholipids with some DNA substructures studied by means of differential scanning calorimetry.

The interaction of dipalmitoylphosphatidylcholine, dipalmitoylphosphatidic acid and dipalmitoylphosphatidylethanolamine with some DNA substructures such as cytidine, uridine, adenosine 5'di- and triphosphate, guanosine 5'mono- and diphosphate, cytidine 5'mono- and triphosphate, uridine 5'mono- and triphosphate and inosine 5'monophosphate was studied with differential scanning calorimetry. The dependence of pretransition and main transition temperatures and the enthalpy of main transition on the molecular characteristics of the interacting molecular species was calculated by stepwise regression analysis. Nucleosides and nucleotides increased the main transition temperature and peak half width of phospholipids and they decreased the enthalpy of main transition proving the existence of interaction between phospholipids and DNA substructures. Calculation proved that the interaction is mainly of hydrophilic character but the involvement of hydrophobic forces or steric conditions cannot be ruled out.     

Thermodynamic parameters of the interaction between Co(II) bovine carbonic anhydrase and anionic inhibitors.

The pH dependence of the apparent affinity constants of perchlorate for cobalt(II)bovine carbonic anhydrase II has been measured by electronic absorption spectroscopy. The obtained data have been analyzed in terms of the ionization of two acidic groups of CoBCAII, and the affinity of perchlorate for the two water-containing species of the enzyme have been estimated. Furthermore, the affinity constants of nitrate, perchlorate, and azide for CoBCAII in the temperature range 5 degrees C-30 degrees C have been determined by spectrophotometric titrations at pH 7. The affinity constants for these ligands decrease with increasing temperatures. The temperature dependence of binding was used to estimate the enthalpy and entropy parameters for the formation of the corresponding 1:1 adducts. The obtained results indicate that binding of these anions to the cobalt enzyme is an enthalpy driven process which is opposed by a moderate entropy change.     

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.