Binding Of Transition State Analogue Research Articles

Decoupling of catalysis and transition state analog binding from mutations throughout a phosphatase revealed by high-throughput enzymology

Using high-throughput microfluidic enzyme kinetics (HT-MEK), we measured over 9,000 inhibition curves detailing impacts of 1,004 single-site mutations throughout the alkaline phosphatase PafA on binding affinity for two transition state analogs (TSAs), vanadate and tungstate. As predicted by catalytic models invoking transition state complementary, mutations to active site and active-site-contacting residues had highly similar impacts on catalysis and TSA binding. Unexpectedly, most mutations to more distal residues that reduced catalysis had little or no impact on TSA binding and many even increased tungstate affinity. These disparate effects can be accounted for by a model in which distal mutations alter the enzyme's conformational landscape, increasing the occupancy of microstates that are catalytically less effective but better able to accommodate larger transition state analogs. In support of this ensemble model, glycine substitutions (rather than valine) were more likely to increase tungstate affinity (but not more likely to impact catalysis), presumably due to increased conformational flexibility that allows previously disfavored microstates to increase in occupancy. These results indicate that residues throughout an enzyme provide specificity for the transition state and discriminate against analogs that are larger only by tenths of an Ångström. Thus, engineering enzymes that rival the most powerful natural enzymes will likely require consideration of distal residues that shape the enzyme's conformational landscape and fine-tune active-site residues. Biologically, the evolution of extensive communication between the active site and remote residues to aid catalysis may have provided the foundation for allostery to make it a highly evolvable trait.

Genetic resistance to purine nucleoside phosphorylase inhibition in Plasmodium falciparum

Plasmodium falciparum causes the most lethal form of human malaria and is a global health concern. The parasite responds to antimalarial therapies by developing drug resistance. The continuous development of new antimalarials with novel mechanisms of action is a priority for drug combination therapies. The use of transition-state analog inhibitors to block essential steps in purine salvage has been proposed as a new antimalarial approach. Mutations that reduce transition-state analog binding are also expected to reduce the essential catalytic function of the target. We have previously reported that inhibition of host and P. falciparum purine nucleoside phosphorylase (PfPNP) by DADMe-Immucillin-G (DADMe-ImmG) causes purine starvation and parasite death in vitro and in primate infection models. P. falciparum cultured under incremental DADMe-ImmG drug pressure initially exhibited increased PfPNP gene copy number and protein expression. At increased drug pressure, additional PfPNP gene copies appeared with point mutations at catalytic site residues involved in drug binding. Mutant PfPNPs from resistant clones demonstrated reduced affinity for DADMe-ImmG, but also reduced catalytic efficiency. The catalytic defects were partially overcome by gene amplification in the region expressing PfPNP. Crystal structures of native and mutated PfPNPs demonstrate altered catalytic site contacts to DADMe-ImmG. Both point mutations and gene amplification are required to overcome purine starvation induced by DADMe-ImmG. Resistance developed slowly, over 136 generations (2136 clonal selection). Transition-state analog inhibitors against PfPNP are slow to induce resistance and may have promise in malaria therapy.

Heat Capacity Changes for Transition-State Analogue Binding and Catalysis with Human 5'-Methylthioadenosine Phosphorylase.

Human 5'-methylthioadenosine phosphorylase (MTAP) catalyzes the phosphorolysis of 5'-methylthioadenosine (MTA). Its action regulates cellular MTA and links polyamine synthesis to S-adenosylmethionine (AdoMet) salvage. Transition state analogues with picomolar dissociation constants bind to MTAP in an entropically driven process at physiological temperatures, suggesting increased hydrophobic character or dynamic structure for the complexes. Inhibitor binding exhibits a negative heat capacity change (-ΔCp), and thus the changes in enthalpy and entropy upon binding are strongly temperature-dependent. The ΔCp of inhibitor binding by isothermal titration calorimetry does not follow conventional trends and is contrary to that expected from the hydrophobic effect. Thus, ligands of increasing hydrophobicity bind with increasing values of ΔCp. Crystal structures of MTAP complexed to transition-state analogues MT-DADMe-ImmA, BT-DADMe-ImmA, PrT-ImmA, and a substrate analogue, MT-tubercidin, reveal similar active site contacts and overall protein structural parameters, despite large differences in ΔCp for binding. In addition, ΔCp values are not correlated with Kd values. Temperature dependence of presteady state kinetics revealed the chemical step for the MTAP reaction to have a negative heat capacity for transition state formation (-ΔCp‡). A comparison of the ΔCp‡ for MTAP presteady state chemistry and ΔCp for inhibitor binding revealed those transition-state analogues most structurally and thermodynamically similar to the transition state. Molecular dynamics simulations of MTAP apoenzyme and complexes with MT-DADMe-ImmA and MT-tubercidin show small, but increased dynamic motion in the inhibited complexes. Variable temperature CD spectroscopy studies for MTAP-inhibitor complexes indicate remarkable protein thermal stability (to Tm = 99 °C) in complexes with transition-state analogues.

Transition States and Transition State Analogue Interactions with Enzymes

Enzymatic transition states have lifetimes of a few femtoseconds (fs). Computational analysis of enzyme motions leading to transition state formation suggests that local catalytic site motions on the fs time scale provide the mechanism to locate transition states. An experimental test of protein fs motion and its relation to transition state formation can be provided by isotopically heavy proteins. Heavy enzymes have predictable mass-altered bond vibration states without altered electrostatic properties, according to the Born-Oppenheimer approximation. On-enzyme chemistry is slowed in most heavy proteins, consistent with altered protein bond frequencies slowing the search for the transition state. In other heavy enzymes, structural changes involved in reactant binding and release are also influenced. Slow protein motions associated with substrate binding and catalytic site preorganization are essential to allow the subsequent fs motions to locate the transition state and to facilitate the efficient release of products. In the catalytically competent geometry, local groups move in stochastic atomic motion on the fs time scale, within transition state-accessible conformations created by slower protein motions. The fs time scale for the transition state motions does not permit thermodynamic equilibrium between the transition state and stable enzyme states. Isotopically heavy enzymes provide a diagnostic tool for fast coupled protein motions to transition state formation and mass-dependent conformational changes. The binding of transition state analogue inhibitors is the opposite in catalytic time scale to formation of the transition state but is related by similar geometries of the enzyme-transition state and enzyme-inhibitor interactions. While enzymatic transition states have lifetimes as short as 10(-15) s, transition state analogues can bind tightly to enzymes with release rates greater than 10(3) s. Tight-binding transition state analogues stabilize the rare but evolved enzymatic geometry to form the transition state. Evolution to efficient catalysis optimized this geometry and its stabilization by a transition state mimic results in tight binding. Release rates of transition state analogues are orders of magnitude slower than product release in normal catalytic function. During catalysis, product release is facilitated by altered chemistry. Compared to the weak associations found in Michaelis complexes, transition state analogues involve strong interactions related to those in the transition state. Optimum binding of transition state analogues occurs when the complex retains the system motions intrinsic to transition state formation. Conserved dynamic motion retains the entropic components of inhibitor complexes, improving the thermodynamics of analogue binding.

Active site and remote contributions to catalysis in methylthioadenosine nucleosidases.

5'-Methylthioadenosine/S-adenosyl-l-homocysteine nucleosidases (MTANs) catalyze the hydrolysis of 5'-methylthioadenosine to adenine and 5-methylthioribose. The amino acid sequences of the MTANs from Vibrio cholerae (VcMTAN) and Escherichia coli (EcMTAN) are 60% identical and 75% similar. Protein structure folds and kinetic properties are similar. However, binding of transition-state analogues is dominated by favorable entropy in VcMTAN and by enthalpy in EcMTAN. Catalytic sites of VcMTAN and EcMTAN in contact with reactants differ by two residues; Ala113 and Val153 in VcMTAN are Pro113 and Ile152, respectively, in EcMTAN. We mutated the VcMTAN catalytic site residues to match those of EcMTAN in anticipation of altering its properties toward EcMTAN. Inhibition of VcMTAN by transition-state analogues required filling both active sites of the homodimer. However, in the Val153Ile mutant or double mutants, transition-state analogue binding at one site caused complete inhibition. Therefore, a single amino acid, Val153, alters the catalytic site cooperativity in VcMTAN. The transition-state analogue affinity and thermodynamics in mutant VcMTAN became even more unlike those of EcMTAN, the opposite of expectations from catalytic site similarity; thus, catalytic site contacts in VcMTAN are unable to recapitulate the properties of EcMTAN. X-ray crystal structures of EcMTAN, VcMTAN, and a multiple-site mutant of VcMTAN most closely resembling EcMTAN in catalytic site contacts show no major protein conformational differences. The overall protein architectures of these closely related proteins are implicated in contributing to the catalytic site differences.

Distortional binding of transition state analogs to human purine nucleoside phosphorylase probed by magic angle spinning solid-state NMR.

Transition state analogs mimic the geometry and electronics of the transition state of enzymatic reactions. These molecules bind to the active site of the enzyme much tighter than substrate and are powerful noncovalent inhibitors. Immucillin-H (ImmH) and 4'-deaza-1'-aza-2'-deoxy-9-methylene Immucillin-H (DADMe-ImmH) are picomolar inhibitors of human purine nucleoside phosphorylase (hPNP). Although both molecules are electronically similar to the oxocarbenium-like dissociative hPNP transition state, DADMe-ImmH is more potent than ImmH. DADMe-ImmH captures more of the transition state binding energy by virtue of being a closer geometric match to the hPNP transition state than ImmH. A consequence of these similarities is that the active site of hPNP exerts greater distortional forces on ImmH than on DADMe-ImmH to "achieve" the hPNP transition state geometry. By using magic angle spinning solid-state NMR to investigate stable isotope-labeled ImmH and DADMe-ImmH, we have explored the difference in distortional binding of these two inhibitors to hPNP. High-precision determinations of internuclear distances from NMR recoupling techniques, rotational echo double resonance, and rotational resonance, have provided unprecedented atomistic insight into the geometric changes that occur upon binding of transition state analogs. We conclude that hPNP stabilizes conformations of these chemically distinct analogs having distances between the cation and leaving groups resembling those of the known 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 Å 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.

Tight binding of transition-state analogues to a peptidyl-aminoacyl-L/D-isomerase from frog skin.

d-Amino acids in peptide linkage are a noteworthy exception from the universal homochirality of proteins and peptides.[1] The first animal peptide that was found to contain a d-amino acid as the second residue was dermorphin, isolated from the skin of a South American tree frog.[2] This peptide binds with high affinity to μ-opiate receptors. The d-residue in this and related amphibian opioid peptides is essential for the biological function, whereas the all-L-isomers lack activity.

Enzymatic Transition States, Transition-State Analogs, Dynamics, Thermodynamics, and Lifetimes

Experimental analysis of enzymatic transition-state structures uses kinetic isotope effects (KIEs) to report on bonding and geometry differences between reactants and the transition state. Computational correlation of experimental values with chemical models permits three-dimensional geometric and electrostatic assignment of transition states formed at enzymatic catalytic sites. The combination of experimental and computational access to transition-state information permits (a) the design of transition-state analogs as powerful enzymatic inhibitors, (b) exploration of protein features linked to transition-state structure, (c) analysis of ensemble atomic motions involved in achieving the transition state, (d) transition-state lifetimes, and (e) separation of ground-state (Michaelis complexes) from transition-state effects. Transition-state analogs with picomolar dissociation constants have been achieved for several enzymatic targets. Transition states of closely related isozymes indicate that the protein's dynamic architecture is linked to transition-state structure. Fast dynamic motions in catalytic sites are linked to transition-state generation. Enzymatic transition states have lifetimes of femtoseconds, the lifetime of bond vibrations. Binding isotope effects (BIEs) reveal relative reactant and transition-state analog binding distortion for comparison with actual transition states.

Importance of Arg-599 of β-galactosidase (Escherichia coli) as an anchor for the open conformations of Phe-601 and the active-site loop

Structural and kinetic data show that Arg-599 of β-galactosidase plays an important role in anchoring the "open" conformations of both Phe-601 and an active-site loop (residues 794-803). When alanine was substituted for Arg-599, the conformations of Phe-601 and the loop shifted towards the "closed" positions because interactions with the guanidinium side chain were lost. Also, Phe-601, the loop, and Na+, which is ligated by the backbone carbonyl of Phe-601, lost structural order, as indicated by large B-factors. IPTG, a substrate analog, restored the conformations of Phe-601 and the loop of R599A-β-galactosidase to the open state found with IPTG-complexed native enzyme and partially reinstated order. ᴅ-Galactonolactone, a transition state analog, restored the closed conformations of R599A-β-galactosidase to those found with ᴅ-galactonolactone-complexed native enzyme and completely re-established the order. Substrates and substrate analogs bound R599A-β-galactosidase with less affinity because the closed conformation does not allow substrate binding and extra energy is required for Phe-601 and the loop to open. In contrast, transition state analog binding, which occurs best when the loop is closed, was several-fold better. The higher energy level of the enzyme•substrate complex and the lower energy level of the first transition state means that less activation energy is needed to form the first transition state and thus the rate of the first catalytic step (k2) increased substantially. The rate of the second catalytic step (k3) decreased, likely because the covalent form is more stabilized than the second transition state when Phe-601 and the loop are closed. The importance of the guanidinium group of Arg-599 was confirmed by restoration of conformation, order, and activity by guanidinium ions.

Conformational States of Human Purine Nucleoside Phosphorylase at Rest, at Work, and with Transition State Analogues

Human purine nucleoside phosphorylase (PNP) is a homotrimer binding tightly to the transition state analogues Immucillin-H (ImmH; K(d) = 56 pM) and DATMe-ImmH-Immucillin-H (DATMe-ImmH; K(d) = 8.6 pM). ImmH binds with a larger entropic penalty than DATMe-ImmH, a chemically more flexible inhibitor. The testable hypothesis is that PNP conformational states are more relaxed (dynamic) with DATMe-ImmH, despite tighter binding than with ImmH. PNP conformations are probed by peptide amide deuterium exchange (HDX) using liquid chromatography high-resolution Fourier transform ion cyclotron resonance mass spectrometry and by sedimentation rates. Catalytically equilibrating Michaelis complexes (PNP.PO(4).inosine <--> PNP.Hx.R-1-P) and inhibited complexes (PNP.PO(4).DATMe-ImmH and PNP.PO(4).ImmH) show protection from HDX at 9, 13, and 15 sites per subunit relative to resting PNP (PNP.PO(4)) in extended incubations. The PNP.PO(4).ImmH complex is more compact (by sedimentation rate) than the other complexes. HDX kinetic analysis of ligand-protected sites corresponds to peptides near the catalytic sites. HDX and sedimentation results establish that PNP protein conformation (dynamic motion) correlates more closely with entropy of binding than with affinity. Catalytically active turnover with saturated substrate sites causes less change in HDX and sedimentation rates than binding of transition state analogues. DATMe-ImmH more closely mimics the transition of human PNP than does ImmH and achieves strong binding interactions at the catalytic site while causing relatively modest alterations of the protein dynamic motion. Transition state analogues causing the most rigid, closed protein conformation are therefore not necessarily the most tightly bound. Close mimics of the transition state are hypothesized to retain enzymatic dynamic motions related to transition state formation.

Enzymatic transition states and dynamic motion in barrier crossing

What are the atomic motions at enzymatic catalytic sites on the timescale of chemical change? Combined experimental and computational chemistry approaches take advantage of transition-state analogs to reveal dynamic motions linked to transition-state formation. QM/MM transition path sampling from reactive complexes provides both temporal and dynamic information for barrier crossing. Fast (femtosecond to picosecond) dynamic motions provide essential links to enzymatic barrier crossing by local or promoting-mode dynamic searches through bond-vibrational space. Transition-state lifetimes are within the femtosecond timescales of bond vibrations and show no manifestations of stabilized, equilibrated complexes. The slow binding and protein conformational changes (microsecond to millisecond) also required for catalysis are temporally decoupled from the fast dynamic motions forming the transition state. According to this view of enzymatic catalysis, transition states are formed by fast, coincident dynamic excursions of catalytic site elements, while the binding of transition-state analogs is the conversion of the dynamic excursions to equilibrated states.

Testing Geometrical Discrimination within an Enzyme Active Site: Constrained Hydrogen Bonding in the Ketosteroid Isomerase Oxyanion Hole

Enzymes are classically proposed to accelerate reactions by binding substrates within active-site environments that are structurally preorganized to optimize binding interactions with reaction transition states rather than ground states. This is a remarkably formidable task considering the limited 0.1-1 A scale of most substrate rearrangements. The flexibility of active-site functional groups along the coordinate of substrate rearrangement, the distance scale on which enzymes can distinguish structural rearrangement, and the energetic significance of discrimination on that scale remain open questions that are fundamental to a basic physical understanding of enzyme active sites and catalysis. We bring together 1.2-1.5 A resolution X-ray crystallography, (1)H and (19)F NMR spectroscopy, quantum mechanical calculations, and transition-state analogue binding measurements to test the distance scale on which noncovalent forces can constrain the structural relaxation or translation of side chains and ligands along a specific coordinate and the energetic consequences of such geometric constraints within the active site of bacterial ketosteroid isomerase (KSI). Our results strongly suggest that packing and binding interactions within the KSI active site can constrain local side-chain reorientation and prevent hydrogen bond shortening by 0.1 A or less. Further, this constraint has substantial energetic effects on ligand binding and stabilization of negative charge within the oxyanion hole. These results provide evidence that subtle geometric effects, indistinguishable in most X-ray crystallographic structures, can have significant energetic consequences and highlight the importance of using synergistic experimental approaches to dissect enzyme function.

Transition-State Interactions Revealed in Purine Nucleoside Phosphorylase by Binding Isotope Effects

The binding of [5‘-3H]inosine to human purine nucleoside phosphorylase results in an equilibrium binding isotope effect (BIE) of 1.5%, and transition state formation causes an intrinsic KIE of 4.7%. These values reflect atomic vibrational distortions in the 5‘-C−H bonds upon formation of the Michaelis complex and transition state. The degree of atomic distortion for catalysis is compared to that for binding of transition state analogues. Similar radiolabeled forms of the transition-state analogues ImmH and DADMe-ImmH gave large 5‘-3H BIEs of 12.6% and 29.2%, respectively. Greater bond distortions occur upon complex formation with transition-state analogues, supporting weaker distortional forces at the transition state than in the formation of complexes with transition-state analogues.

Anopheles gambiaePurine Nucleoside Phosphorylase: Catalysis, Structure, and Inhibition,

The purine salvage pathway of Anopheles gambiae, a mosquito that transmits malaria, has been identified in genome searches on the basis of sequence homology with characterized enzymes. Purine nucleoside phosphorylase (PNP) is a target for the development of therapeutic agents in humans and purine auxotrophs, including malarial parasites. The PNP from Anopheles gambiae (AgPNP) was expressed in Escherichia coli and compared to the PNPs from Homo sapiens (HsPNP) and Plasmodium falciparum (PfPNP). AgPNP has kcat values of 54 and 41 s-1 for 2'-deoxyinosine and inosine, its preferred substrates, and 1.0 s-1 for guanosine. However, the chemical step is fast for AgPNP at 226 s-1 for guanosine in pre-steady-state studies. 5'-Deaza-1'-aza-2'-deoxy-1'-(9-methylene)-Immucillin-H (DADMe-ImmH) is a transition-state mimic for a 2'-deoxyinosine ribocation with a fully dissociated N-ribosidic bond and is a slow-onset, tight-binding inhibitor with a dissociation constant of 3.5 pM. This is the tightest-binding inhibitor known for any PNP, with a remarkable Km/Ki* of 5.4 x 10(7), and is consistent with enzymatic transition state predictions of enhanced transition-state analogue binding in enzymes with enhanced catalytic efficiency. Deoxyguanosine is a weaker substrate than deoxyinosine, and DADMe-Immucillin-G is less tightly bound than DADMe-ImmH, with a dissociation constant of 23 pM for AgPNP as compared to 7 pM for HsPNP. The crystal structure of AgPNP was determined in complex with DADMe-ImmH and phosphate to a resolution of 2.2 A to reveal the differences in substrate and inhibitor specificity. The distance from the N1' cation to the phosphate O4 anion is shorter in the AgPNP.DADMe-ImmH.PO4 complex than in HsPNP.DADMe-ImmH.SO4, offering one explanation for the stronger inhibitory effect of DADMe-ImmH for AgPNP.

Enzymatic Transition State Theory and Transition State Analogue Design

Enzymatic Transition State Theory and Transition State Analogue Design

Electrostatic Contributions to Binding of Transition State Analogues Can Be Very Different from the Corresponding Contributions to Catalysis: Phenolates Binding to the Oxyanion Hole of Ketosteroid Isomerase

The relationship between binding of transition state analogues (TSAs) and catalysis is an open problem. A recent study of the binding of phenolate TSAs to ketosteroid isomerase (KSI) found a small change in the binding energy with a change in charge delocalization of the TSAs. This has been taken as proof that electrostatic effects do not contribute in a major way to catalysis. Here we reanalyze the relationship between the binding of the TSAs and the chemical catalysis by KSI as well as the binding of the transition state (TS), by computer simulation approaches. Since the simulations reproduce the relevant experimental results, they can be used to quantify the different contributions to the observed effects. It is found that the binding of the TSA and the chemical catalysis represent different thermodynamic cycles with very different electrostatic contributions. While the binding of the TSA involves a small electrostatic contribution, the chemical catalysis involves a charge transfer process and a major electrostatic contribution due to the preorganization of the active site. Furthermore, it is found that the electrostatic preorganization contributions to the binding of the enolate intermediate of KSI and the TS are much larger than the corresponding effect for the binding of the TSAs. This reflects the dependence of the preorganization on the orientation of the nonpolar form of the TSAs relative to the oxyanion hole. It seems to us that this work provides an excellent example of the need for computational studies in analyzing key experimental findings about enzyme catalysis.

Structural Basis of the Transition-state Stabilization in Antibody-catalyzed Hydrolysis

The catalytic antibody 6D9, which was raised against a transition-state analogue (TSA), catalyzes the hydrolysis of a non-bioactive chloramphenicol monoester to generate chloramphenicol. It has been shown that 6D9 utilizes the binding affinity in the catalysis; the differential affinity of the TSA relative to the substrate is equal to the rate enhancement. To reveal the recognition mechanism of 6D9 for the TSA and the substrate, we performed NMR analysis of the Fv fragment of 6D9 (6D9-Fv), together with site-directed mutagenesis and stopped-flow kinetic analyses. Among six 6D9-Fv mutants, Y58(H)A and W100i(H)A displayed significant reductions in their affinities to the TSA, while their substrate-binding affinities were identical with that of the wild-type 6D9-Fv. The stopped-flow kinetic studies revealed that the TSA binding to 6D9-Fv occurred by an induced-fit mechanism. In contrast, no induced-fit type of TSA-binding mechanism was observed for Y58(H)A and W100i(H)A. From NMR experiments, we identified the residues with chemical shifts that were perturbed by the ligand-binding. The residues affected by the TSA binding were located on the TSA-binding site determined by the X-ray study, and on the regions far from the binding site. On the other hand, the residues affected by the substrate binding were localized on the TSA-binding site. As for W100i(H)A, no residue other than those in the binding site was affected by the ligand binding. On the basis of these results and the crystal structure, we concluded that the TSA binding induced a conformational change involving the formation of aromatic–aromatic interactions and a hydrogen bond. These interactions can account for the differential affinity for the TSA relative to the substrate. W100i(H) probably plays an important role in inducing the conformational changes. The present NMR studies have enabled us to visualize the concept of transition-state stabilization in enzymatic catalysis, in which the transition-state contacts are better than those of the substrate.

Origin of Tight Binding of a Near-Perfect Transition-State Analogue by Cytidine Deaminase: Implications for Enzyme Catalysis

Cytidine deaminase (CDA) is a zinc metalloenzyme that catalyzes the hydrolytic deamination of cytidine to uridine. Zebularine (ZEB) binds to CDA, and the binding process leads to a near-perfect transition-state analogue (TSA) inhibitor at the active site with an estimated K(i) value of 1.2 x 10(-)(12) M. The interaction of CDA with the TSA inhibitor has become a paradigm for studying the tight TSA binding by enzymes. The formation of the TSA is catalyzed by CDA by a mechanism that is similar to the formation of the tetrahedral intermediate during the CDA-catalyzed reaction (i.e., through the nucleophilic attack of a Zn-hydroxide group on C(4)). It is believed that the TSA formed at the active site is zebularine 3,4-hydrate. In this paper, it is shown from QM/MM molecular dynamics and free energy simulations that zebularine 3,4-hydrate may in fact be unstable in the enzyme and that a proton transfer from the Zn-hydroxide group to Glu-104 during the nucleophilic attack could be responsible for the very high affinity. The nucleophilic attack by the Zn-hydroxide on C(4) is found to be concerted with two proton transfers. Such concerted process allows the TSA, an alkoxide-like inhibitor, to be stabilized through a mechanism that is similar to the transition-state stabilization in the general acid-base catalysis. It is suggested that the proton transfer from the Zn-hydroxide to Glu-104, which is required to generate the general acid for protonating the leaving ammonia, may play an important role in lowering the activation barrier during the catalysis.

A transition-state analogue reduces protein dynamics in hypoxanthine-guanine phosphoribosyltransferase.

Hypoxanthine-guanine phosphoribosyltransferase (HGPRT) is the key enzyme in purine base salvage in humans and in purine auxotrophs, including Plasmodium falciparum, the leading cause of malaria. Hydrogen/deuterium (H/D) exchange into amide bonds, quantitated by on-line HPLC and mass spectrometry, has been used to compare the dynamic and conformational properties of human HGPRT alone, the HGPRT-GMP-Mg(2+) complex, the HGPRT-IMP-MgPPi <==> HGPRT-Hx-MgPRPP equilibrating mixture, and the transition-state analogue complex HGPRT-ImmGP-MgPPi. The rate and extent of H/D exchange of 26 peptic peptides, spanning 91% of the primary structure, have been monitored. Human HGPRT has 207 amide H/D exchange sites. After 1 h in D2O, HGPRT alone exchanges 160, HGPRT-GMP-Mg(2+) exchanges 154, the equilibrium complex exchanges 139, and the transition-state analogue complex exchanges 126 of these amide protons. H/D exchange rates are correlated with structure for peptides in (1) catalytic site loops, (2) a connected peptide of the subunit interface of the tetramer, and (3) a loop buried in the catalytic site. Structural properties related to H/D exchange are defined from crystallographic studies of the HGPRT-GMP-Mg(2+) and HGPRT-ImmGP-MgPPi complexes. Transition-state analogue binding strengthens the interaction between subunits and tightens the catalytic site loops. The solvent exchange dynamics in specific peptides correlates with hydrogen bond patterns, solvent access, crystallographic B-factors, and ligand exchange rates. Solvent exchange reveals loop dynamics in the free enzyme, Michaelis complexes, and the complex with the bound transition-state analogue. Proton transfer paths, rather than dynamic motion, are required to explain exchange into a buried catalytic site peptide in the complex with the bound transition-state analogue.