Transition State Analog Complex Research Articles

Serine proteases: An ab initio molecular dynamics study

In serine proteases (SP's), the H-bond between His-57 and Asp-102, and that between Gly-193 and the transition state intermediate play a crucial role for enzymatic function. To shed light on the nature of these interactions, we have carried out ab initio molecular dynamics simulations on complexes representing adducts between the reaction intermediate and elastase (one protein belonging to the SP family). Our calculations indicate the presence of a low--barrier H-bond between His-57 and Asp-102, in complete agreement with NMR experiments on enzyme--transition state analog complexes. Comparison with an ab initio molecular dynamics simulation on a model of the substrate--enzyme adduct indicates that the Gly-193--induced strong stabilization of the intermediate is accomplished by charge/dipole interactions and not by H-bonding as previously suggested. Inclusion of the protein electric field in the calculations does not affect significantly the charge distribution.

Free radical-induced inactivation of creatine kinase: influence on the octameric and dimeric states of the mitochondrial enzyme (Mib-CK)

Free radicals of X-ray-induced water radiolysis, either directly or indirectly via their reaction products, reduce the activity of both dimeric cytoplasmic muscle-type creatine kinase (MM-CK) and octameric mitochondrial creatine kinase (Mi-CK) to virtually zero. Similarly values of the characteristic D(37)-dose of enzyme inactivation (dose required to reduce enzyme activity to 37%) were found for the two isoenzymes of CK under identical conditions. Octamer stability was not significantly affected within the dose range considered. However, both the dissociation of octamers into dimers by a transition-state analogue complex (TSAC), and the reassociation of the dimers into octamers, showed dose-dependent reduction. Binding of the TSAC to the active centre was found to protect the enzyme against inactivation by free radicals. No protection was observed for the radiation-induced decrease of the endogenous tryptophan fluorescence. The experimental results are in line with the following interpretation: (i) the reduction of Mi(b)-CK dimer association is due to free radical-induced modification of Trp-264, situated at the dimer/dimer interface; (ii) the active-site Trp-223 is not a prime target for free radicals and is not involved in the inactivation of the enzyme; (iii) the inhibition of TSAC-induced dissociation of Mi(b)-CK, like enzyme inactivation, is primarily due to a modification of the active-site Cys-278.

Impaired transition state complementarity in the hydrolysis of O-arylphosphorothioates by protein-tyrosine phosphatases.

The hydrolysis of O-arylphosphorothioates by protein-tyrosine phosphatases (PTPases) was studied with the aim of providing a mechanistic framework for the reactions of this important class of substrate analogues. O-arylphosphorothioates are hydrolyzed 2 to 3 orders of magnitude slower than O-aryl phosphates by PTPases. This is in contrast to the solution reaction where phosphorothioates display 10-60-fold higher reactivity than the corresponding oxygen analogues. Kinetic analyses suggest that PTPases utilize the same active site and similar kinetic and chemical mechanisms for the hydrolysis of O-arylphosphorothioates and O-aryl phosphates. Thio substitution has no effect on the affinity of substrate or product for the PTPases. Bronsted analyses suggest that like the PTPase-catalyzed phosphoryl transfer reaction the transition state for the PTPase-catalyzed thiophosphoryl transfer is highly dissociative, similar to that of the corresponding solution reaction. The side chain of the active-site Arg residue forms a bidentate hydrogen bond with two of the terminal phosphate oxygens in the ground state and two of the equatorial oxygens in a transition state analog complex with vanadate [Denu et al. (1996) Proc. Natl. Acad. Sci. USA 93, 2493-2498; Zhang, M. et al. (1997) Biochemistry 36, 15-23; Pannifer et al. (1998) J. Biol. Chem. 273, 10454-10462]. Replacement of the active-site Arg409 in the Yersinia PTPase by a Lys reduces the thio effect by 54-fold, consistent with direct interaction and demonstrating strong energetic coupling between Arg409 and the phosphoryl oxygens in the transition state. These results suggest that the large thio effect observed in the PTPase reaction is the result of inability to achieve precise transition state complementarity in the enzyme active site with the larger sulfur substitution.

Detection of nucleotide- and F-actin-induced movements in the switch II helix of the skeletal myosin using its differential oxidative cleavage mediated by an iron-EDTA complex disulfide-linked to the strong actin binding site.

We have synthesized the mixed disulfide, S-(2-nitro-5-thiobenzoic acid) cysteaminyl-EDTA, using a rapid procedure and water-soluble chemistry. Its disulfide-thiol exchange reaction with rabbit myosin subfragment-1 (S-1), analyzed by spectrophotometry, ATPase assays, and peptide mapping, led to the incorporation of the cysteaminyl-EDTA group into only Cys 540 on the heavy chain and into the unique cysteine on the alkali light chains. The former thiol, residing in the strong actin binding site, reacted at a much faster rate with a concomitant 3-fold decrease in the V(max) for acto-S-1 ATPase but without change in the essential enzymatic functions of S-1. Upon chelation of Fe(3+) ions to the Cys 540-bound EDTA and incubation of the S-1 derivative-Fe complex with ascorbic acid at pH 7.5, the 95 kDa heavy chain underwent a conformation-dependent, single-cut oxidative fragmentation within 5-15 A of Cys 540. Three pairs of fragments were formed which, after specific fluorescent labeling and SDS-PAGE, could be positioned along the heavy chain sequence as 68 kDa-26 kDa, 62 kDa-32 kDa, and 54 kDa-40 kDa. Densitometric measurements revealed that the yield of the 54 kDa-40 kDa pair of bands, but not that for the two other pairs, was very sensitive to S-1 binding to nucleotides or phosphate analogues as well as to F-actin. In binary complexes, all the former ligands specifically lowered the yield to 40% of S-1 alone, roughly in the following order: ADP = AMP-PNP > ATP = ADP.AlF(4) > ADP.BeF(x)() > PP(i). By contrast, rigor binding to F-actin increased the yield to 130%. In the ternary acto-S-1-ADP complex, the yield was again reduced to 80%, and it fell to 25% in acto-S-1-ADP.AlF(4), the putative transition state analogue complex of the acto-S-1 ATPase. These different quantitative changes reflect distinct ligand-induced conformations of the secondary structure element whose scission generates the 54 kDa-40 kDa species. According to the S-1 crystal structure, this element could be unambiguously assigned to the switch II helix (residues 475-507) whose N-terminus lies 14.2 A from Cys 540 and would include the ligand-responsive cleavage site. This motif is thought to be crucial for the transmission of sub-nanometer structural changes at the ATPase site to both the actin site and the lever arm domain during energy transduction. Our study illustrates this novel, actin site-specific chemical proteolysis of S-1 as a direct probe of the switch II helix conformational transitions in solution most likely associated with the skeletal cross-bridge cycle.

Linear-scaling quantum mechanical calculations of biological molecules: The divide-and-conquer approach

The divide-and-conquer technique for linear-scaling quantum mechanical calculations is reviewed. The method divides a large system into many subsystems, determines the density matrix of each subsystem separately, and sums the corresponding subsystem contributions to obtain the total density matrix and the energy of the system. There is a uniform chemical potential to allow transfer of electrons between subsystems and to insure the normalization of the electron density. The implementation of the method for semiempirical quantum chemistry Hamiltonians is described. The review describes the application to the study of the catalytic mechanisms of cytidine deaminase, an enzyme which accelerates the rate of hydrolytic deamination of cytidine to uridine. The linear-scaling quantum mechanical calculations determined the active species of the ground-state complex and the structure of the reaction transition-state analog complex.

Structural Changes of Creatine Kinase upon Substrate Binding

Small-angle x-ray scattering was used to investigate structural changes upon binding of individual substrates or a transition state analog complex (TSAC; Mg-ADP, creatine, and KNO 3) to creatine kinase (CK) isoenzymes (dimeric muscle-type (M)-CK and octameric mitochondrial (Mi)-CK) and monomeric arginine kinase (AK). Considerable changes in the shape and the size of the molecules occurred upon binding of Mg-nucleotide or TSAC. The radius of gyration of Mi-CK was reduced from 55.6 Å (free enzyme) to 48.9 Å (enzyme plus Mg-ATP) and to 48.2 Å (enzyme plus TSAC). M-CK showed similar changes from 28.0 Å (free enzyme) to 25.6 Å (enzyme plus Mg-ATP) and to 25.5 Å (enzyme plus TSAC). Creatine alone did not lead to significant changes in the radii of gyration, nor did free ATP or ADP. AK also showed a change of the radius of gyration from 21.5 Å (free enzyme) to 19.7 Å (enzyme plus Mg-ATP), whereas with arginine alone only a minor change could be observed. The primary change in structure as seen with monomeric AK seems to be a Mg-nucleotide-induced domain movement relative to each other, whereas the effect of substrate may be of local order only. In CK, however, additional movements have to be involved.

Transition state structure of arginine kinase: implications for catalysis of bimolecular reactions.

Arginine kinase belongs to the family of enzymes, including creatine kinase, that catalyze the buffering of ATP in cells with fluctuating energy requirements and that has been a paradigm for classical enzymological studies. The 1.86-A resolution structure of its transition-state analog complex, reported here, reveals its active site and offers direct evidence for the importance of precise substrate alignment in the catalysis of bimolecular reactions, in contrast to the unimolecular reactions studied previously. In the transition-state analog complex studied here, a nitrate mimics the planar gamma-phosphoryl during associative in-line transfer between ATP and arginine. The active site is unperturbed, and the reactants are not constrained covalently as in a bisubstrate complex, so it is possible to measure how precisely they are pre-aligned by the enzyme. Alignment is exquisite. Entropic effects may contribute to catalysis, but the lone-pair orbitals are also aligned close enough to their optimal trajectories for orbital steering to be a factor during nucleophilic attack. The structure suggests that polarization, strain toward the transition state, and acid-base catalysis also contribute, but, in contrast to unimolecular enzyme reactions, their role appears to be secondary to substrate alignment in this bimolecular reaction.

Mitochondrial Creatine Kinase Is a Prime Target of Peroxynitrite-induced Modification and Inactivation

The reaction of peroxynitrite (PN) with sarcomeric mitochondrial creatine kinase (Mib-CK; EC 2.7.3.2) was observed at different stages of complexity (i) with purified Mi-CK, (ii) with enzyme bound on isolated mitoplasts, and (iii) within intact respiring mitochondria. Creatine-stimulated respiration was abolished by PN concentrations likely to be physiological and far before the respiratory chain itself was affected, thus demonstrating that Mi-CK is a prime target for inactivation by PN in intact mitochondria. The inactivation by PN of Mi-CK was reversed by 22% with 2-mercaptoethanol. More remarkable protective effects were noticed with the full set of CK substrates, e.g. 30 and 50% protection with MgATP plus creatine and MgADP plus phosphocreatine, respectively, but not with each substrate alone. These data indicate an involvement of the active-site Cys-278 residue of Mi-CK in this process. Furthermore, changes in endogenous tryptophan fluorescence intensity and spectral changes after reaction of Mi-CK with PN suggest additional modifications of Trp and Tyr residues. PN-inactivated Mi-CK can no longer be efficiently converted into dimers by incubation with reagents inducing a transition state analog complex at the active site. Thus, obviously, upon reaction of octameric Mi-CK with PN, the octamer-dimer equilibrium of Mi-CK is also affected. The consequences for cellular energy homeostasis and calcium handling are discussed.

Structure of the Ribonuclease·Uridine−Vanadate Transition State Analogue Complex by Raman Difference Spectroscopy: Mechanistic Implications

Raman difference spectroscopy is used to assess changes in the internal bonding of the oxygens of a ⪫VO2- group when that group is incorporated into a complex also involving RNase A, uridine, and a water molecule (the RNase/UVO2/H2O complex). We find that the strengths of the nonbridging VO bonds are decreased by 0.055 vu and their bond lengths are decreased 0.012 Å, based on the stretching frequency changes of nonbridging VO bonds upon formation of the enzymic RNase/UVO2/H2O complex from the cyclic vanadate diester in solution. The bond lengths are 1.638 Å for the solution complex and 1.650 Å for the enzymic complex. The values found for the bond lengths are about 0.15 Å shorter than those found previously in crystallographic studies. Assuming the RNase/UVO2/H2O adduct is a reasonably good transition state analogue, our Raman results suggest that the RNase-catalyzed hydrolysis of uridine 2‘,3‘-cyclic phosphate proceeds via an SN2-like process. The process also may involve a small associative charactor, since the summed bond strength of nonbridging PO bonds is reduced by only 0.11−0.22 vu in the transition state compared with the ground state, which means the summed bond strength of the PO bonds of the entering and leaving groups is correspondingly increased.

Low Barrier Hydrogen Bond Is Absent in the Catalytic Triads in the Ground State but Is Present in a Transition-state Complex in the Prolyl Oligopeptidase Family of Serine Proteases

High frequency proton NMR spectra for two members of the prolyl oligopeptidase class of serine proteases, prolyl oligopeptidase and oligopeptidase B, showed that resonances corresponding to the active center histidine Ndelta1H and Nepsilon2H generally observed in this region, are absent in these enzymes. However, for both enzymes, as well as with the H652A and H652Q active center variants of oligopeptidase B, there are two resonances observed in this region that could be assigned to two protonated histidines with a noncatalytic function. The results indicate that these two histidines participate in strong hydrogen bonds. The absence of resonances pertinent to the active center histidine resonances suggests the absence of a low barrier hydrogen bond between the Asp and His in these two enzymes in their ground states. Addition of the peptide boronic acid t-butoxycarbonyl-(D)Val-Leu-(L)boroArg to oligopeptidase B resulted in potent, slow binding inhibition of the enzyme and the appearance of a new resonance at 15.8 ppm, whose chemical shift is appropriate for a tetrahedral boronate complex and a low barrier hydrogen bond. The results demonstrate important dissimilarities between the active centers of the prolyl oligopeptidase class of serine proteases and the pancreatic and subtilisin classes both in the ground state and in the transition-state analog complexes.

The structure of the cytidine deaminase-product complex provides evidence for efficient proton transfer and ground-state destabilization.

Crystal structures of the cytidine deaminase-uridine product complex prepared either by cocrystallizing enzyme with uridine or by diffusing cytidine into ligand-free crystals show that the product binds as a 4-ketopyrimidine. They reveal four additional features of the catalytic process. (1) A water molecule bound to a site previously observed to bind the incoming 4-NH2 group represents the site for the leaving ammonia molecule. The conserved Pro 128 accommodates both moieties by orienting the carbonyl group of the previous residue. (2) The Glu 104 carboxylate group rotates from its hydrogen bond to the O4 hydroxyl group in transition-state analog complexes, forming a new hydrogen bond to the leaving group moiety. Thus, after stabilizing the hydroxyl group in the transition state, Glu 104 transfers a proton from that group to the leaving amino group, promoting enol-to-keto isomerization of the product. (3) Difference Fourier comparisons with transition-state complexes indicate that the pyrimidine ring rotates toward the zinc by approximately 10 degrees. The active site thus "pulls" the ring and 4-NH2 group in opposite directions during catalysis. To preserve coplanarity of the 4-keto group with the pyrimidine ring, the N1-C1' glycosidic bond bends by approximately 19 degrees out of the ring plane. This distortion may "spring-load" the product complex and promote dissociation. Failure to recognize a similar distortion could explain an earlier crystallographic interpretation of the adenosine deaminase-inosine complex [Wilson, D. K., & Quiocho, F. A. (1994) Nat. Struct. Biol. 1, 691-694]. (4) The Zn-Sgamma132 bond, which lengthens in transition-state complexes, shortens as the O4 atom returns to a state of lower negative charge in the planar product, consistent with our previous proposal that this bond buffers the zinc bond valence, compensating buildup of negative charge on the oxygen nucleophile during catalysis.

Expression, purification from inclusion bodies, and crystal characterization of a transition state analog complex of arginine kinase: a model for studying phosphagen kinases.

Phosphagen kinases catalyze the reversible transfer of a phosphoryl group between guanidino phosphate compounds and ADP, thereby regenerating ATP during bursts of cellular activity. Large quantities of highly pure arginine kinase (EC 2.7.3.3), the phosphagen kinase present in arthropods, have been isolated from E. coli, into which the cDNA for the horseshoe crab enzyme had been cloned. Purification involves size exclusion and anion exchange chromatographies applied in the denatured and refolded states. The recombinant enzyme has been crystallized as a transition state analog complex. Near complete native diffraction data have been collected to 1.86 A resolution. Substitution of a recombinant source for a natural one, improvement in the purification, and data collection at cryo temperatures have all yielded significant improvements in diffraction.

Loop and subdomain movements in the mechanism of Escherichia coli dihydrofolate reductase: crystallographic evidence.

The reaction catalyzed by Escherichia coli dihydrofolate reductase (ecDHFR) cycles through five detectable kinetic intermediates: holoenzyme, Michaelis complex, ternary product complex, tetrahydrofolate (THF) binary complex, and THF.NADPH complex. Isomorphous crystal structures analogous to these five intermediates and to the transition state (as represented by the methotrexate-NADPH complex) have been used to assemble a 2.1 A resolution movie depicting loop and subdomain movements during the catalytic cycle (see Supporting Information). The structures suggest that the M20 loop is predominantly closed over the reactants in the holoenzyme, Michaelis, and transition state complexes. But, during the remainder of the cycle, when nicotinamide is not bound, the loop occludes (protrudes into) the nicotinamide-ribose binding pocket. Upon changing from the closed to the occluded conformation, the central portion of the loop rearranges from beta-sheet to 3(10) helix. The change may occur by way of an irregularly structured open loop conformation, which could transiently admit a water molecule into position to protonate N5 of dihydrofolate. From the Michaelis to the transition state analogue complex, rotation between two halves of ecDHFR, the adenosine binding subdomain and loop subdomain, closes the (p-aminobenzoyl)glutamate (pABG) binding crevice by approximately 0.5 A. Resulting enhancement of contacts with the pABG moiety may stabilize puckering at C6 of the pteridine ring in the transition state. The subdomain rotation is further adjusted by cofactor-induced movements (approximately 0.5 A) of helices B and C, producing a larger pABG cleft in the THF.NADPH analogue complex than in the THF analogue complex. Such movements may explain how THF release is assisted by NADPH binding. Subdomain rotation is not observed in vertebrate DHFR structures, but an analogous loop movement (residues 59-70) appears to similarly adjust the pABG cleft width, suggesting that these movements are important for catalysis. Loop movement, also unobserved in vertebrate DHFR structures, may preferentially weaken NADP+ vs NADPH binding in ecDHFR, an evolutionary adaptation to reduce product inhibition in the NADP+ rich environment of prokaryotes.

Changes of creatine kinase structure upon ligand binding as seen by small-angle scattering

Small-angle X-ray and neutron scattering have been used to investigate structural changes upon binding of individual substrates or a transition state analogue complex (TSAC), consisting of Mg-ADP, creatine and KNO 3 to creatine kinase isoenzymes (dimeric M-CK and octameric Mi-CK) and monomeric arginine kinase (AK). Considerable changes in the shape and the size of the molecules occurred upon binding of Mg-ATP and TSAC, whereas creatine alone had only a small effect. In Mi-CK, the radius of gyration was reduced from 55.6 Å (free enzyme) to 48.9 Å (enzyme + Mg-ATP) and to 48.2 Å (enzyme + TSAC). The experiments performed with M-CK showed similar changes from 28.0 Å (free enzyme) to 25.6 Å (enzyme + Mg-ATP) and to 25.5 Å (enzyme + TSAC). Creatine alone did not lead to significant changes in the radii of gyration, nor did free ATP or ADP. AK showed the same behaviour: a change of the radius of gyration from 21.5 Å (free enzyme) to 19.7 Å (enzyme + MG-ATP), whereas with arginine alone only a minor change could be observed. The primary change in structure as seen with monomeric AK seems to be a magnesium-nucleotide induced domain movement relative to each other, whereas the effect of substrate may be of local order only. In creatine kinase, however, further movements must be involved in the large conformational change.

New Trifluoromethyl Ketones as Potent Inhibitors of Esterases:19F NMR Spectroscopy of Transition State Analog Complexes and Structure–Activity Relationships

A variety of trifluoromethyl ketones (TFMKs) have been studied as inhibitors of the antennal esterases of the Egyptian armywormSpodoptera littoralis.The chemicals behaved as tight slow-binding inhibitors, the β-thio derivatives being the most potent ones, particularly 3-octylthio-1,1,1-trifluoropropan-2-one (OTFP) with an IC50of 0.08 μM. Other TFMKs, containing a propenyl group at the non-polar end of the molecule as in the natural pheromone structure (compounds4, 8and9), were also notably active. Correlation studies of inhibition with lipophilicity (log P) indicated that the optimum log P values for activity of the β-thio compounds are in the range of 3.03–5.11, while a higher lipophilicity (range 5.37-5.89) was required among the devoid of sulfur ketones.19F NMR studies, carried out with OTFP, showed that the inhibitor binds the enzyme in a reversible manner, forming an adduct (probably a hemiacetal) of tetrahedral geometry with the active site of the enzyme. To our knowledge, this is the first time that such a study on a natural esterase extract has been undertaken.

The X-ray Structure of a Transition State Analog Complex Reveals the Molecular Origins of the Catalytic Power and Substrate Specificity of Acetylcholinesterase

The structure of a complex of Torpedo californica acetylcholinesterase with the transition state analog inhibitor m-(N,N,N-trimethylammonio)-2,2,2-trifluoroacetophenone has been solved by X-ray crystallographic methods to 2.8 A resolution. Since the inhibitor binds to the enzyme about 1010-fold more tightly than the substrate acetylcholine, this complex provides a visual accounting of the enzyme−ligand interactions that provide the molecular basis for the catalytic power of acetylcholinesterase. The enzyme owes about 8 kcal mol-1 of the 18 kcal mol-1 of free energy of stabilization of the acylation transition state to interactions of the quaternary ammonium moiety with three water molecules, with the carboxylate side chain of E199, and with the aromatic side chains of W84 and F330. The carbonyl carbon of the trifluoroketone function interacts covalently with S200 of the S200−H440−E327 catalytic triad. The operation of this triad as a general acid−base catalytic network probably provides 3−5 kcal mol-1 of ...

Structure of the Dimeric Ethylene Glycol-Vanadate Complex and Other 1,2-Diol-Vanadate Complexes in Aqueous Solution: Vanadate-Derived Transition-State Analog Complexes of Phosphotransferases

Structure of the Dimeric Ethylene Glycol-Vanadate Complex and Other 1,2-Diol-Vanadate Complexes in Aqueous Solution: Vanadate-Derived Transition-State Analog Complexes of Phosphotransferases

Dimer-dimer interactions in octameric mitochondrial creatine kinase

Mitochondrial creatine kinase (Mi-CK) forms octamers and dimers, which are readily interconvertible in vitro. The kinetic and thermodynamic octamer stability of wild-type and two mutant, octamer-destabilized forms of chicken sarcomeric Mi-CK was investigated at varying temperatures, pHs, and salt and substrate concentrations, in order to identify parameters which might regulate the octamer/dimer ratio in vivo and to assess the nature of octamer-stabilizing interactions. For wild-type Mi-CK, the rate of the transition state analogue complex (TSAC)-induced octamer decay increased with increasing temperature up to 28 degrees C; increasing pH markedly accelerated the decay in a biphasic manner. The substrate-dependent decay data suggest that also the productive enzymatic transition state of Mi-CK induces an octamer-destabilizing conformation. Thermodynamically, the octamers are stabilized by a combination of hydrophobic and polar contributions. Van't Hoff analysis showed that hydrophobic interactions dominate both in the absence of substrates and in the TSAC conformation, since the equilibrium octamer fractions increased with increasing temperatures, in spite of the accelerated decay kinetics. For the Mi-CK mutant E4Q, a similar temperature dependence was found; in contrast, mutant W264C exhibited an inverted temperature dependence, suggesting that hydrophobic interactions might be largely abolished in this mutant. Both the kinetic and the thermodynamic data seem to suggest that the octamer-dimer transitions of Mi-CK might not play a major role in a fast regulation of mitochondrial energy metabolism, but could rather be involved in slow long-term modulations.

Low-Barrier Hydrogen Bonds

Proteins contain mainly weak, conventional hydrogen bonds; however, a few enzymes have low barrier hydrogen bonds (LBHBs) in transition state analog complexes. Hydrogen bonds display variations in physicochemical properties including length, spectroscopic characteristics, and strength. Three classes of hydrogen bonds have been defined – weak (2–8 kcal mol −1 ), strong (10−2 kcal mol −1 ), and very strong (24−40 kcal mol −1 ). In a weak hydrogen bond, the proton is bonded to one heteroatom by a dipolar covalent bond and engages in a weak electrostatic attraction with another heteroatom. In a strong hydrogen bond, or LBHB, the heteroatoms are closer than a van der Waals contact, the covalent bond to the proton is elongated, and the contact between the proton and the second heteroatom is significantly shorter than in a weak hydrogen bond. In a very strong, or single-well hydrogen bond, the heteroatoms are much closer than a van der Waals contact, and the proton is nearly centered between them. LBHBs in proteins and small molecules are characterized by their spectroscopic and thermodynamic properties, deuterium fractionation factors, and crystallographic structures. LBHBs have been identified in transition state analog complexes of a few enzymes and are postulated to stabilize the transition states in catalysis.

Transition-state selectivity for a single hydroxyl group during catalysis by cytidine deaminase.

Cytidine deaminase binds transition-state analog inhibitors approximately 10(7) times more tightly than corresponding 3,4-dihydro analogs containing a proton in place of the 4-hydroxyl group. X-ray crystal structures of complexes with the two matched inhibitors differ only near a "trapped" water molecule in the complex with the 3,4-dihydro analog, where contacts are substantially less favorable than those with the hydroxyl group of the transition-state analog. The hydrogen bond between the hydroxyl group and the Glu 104 carboxylate shortens in that complex, and may become a "low-barrier" hydrogen bond, since at the same time the bond between zinc and the Cys 132 thiolate ligand lengthens. These differences must therefore account for most of the differential binding affinity related to catalysis. Moreover, the trapped water molecule retains some of the binding energy stabilizing the hydroxyl group in the transition-state analog complex. To this extent, the ratio of binding affinities for the two compounds is smaller than the true contribution of the hydroxyl group, a conclusion with significant bearing on interpreting difference free energies derived from substituent effects arising from chemical modification and/or mutagenesis.