Water in the active site of ketosteroid isomerase

Classical molecular dynamics simulations were utilized to investigate the structural and dynamical properties of water in the active site of ketosteroid isomerase (KSI) to provide insight into the role of these water molecules in the enzyme-catalyzed reaction. This reaction is thought to proceed via a dienolate intermediate that is stabilized by hydrogen bonding with residues Tyr16 and Asp103. A comparative study was performed for the wild-type (WT) KSI and the Y16F, Y16S, and Y16F/Y32F/Y57F (FFF) mutants. These systems were studied with three different bound ligands: equilenin, which is an intermediate analog, and the intermediate states of two steroid substrates. Several distinct water occupation sites were identified in the active site of KSI for the WT and mutant systems. Three additional sites were identified in the Y16S mutant that were not occupied in WT KSI or the other mutants studied. The number of water molecules directly hydrogen bonded to the ligand oxygen was approximately two in the Y16S mutant and one in the Y16F and FFF mutants, with intermittent hydrogen bonding of one water molecule in WT KSI. The molecular dynamics trajectories of the Y16F and FFF mutants reproduced the small conformational changes of residue 16 observed in the crystal structures of these two mutants. Quantum mechanical/molecular mechanical calculations of (1)H NMR chemical shifts of the protons in the active site hydrogen-bonding network suggest that the presence of water in the active site does not prevent the formation of short hydrogen bonds with far-downfield chemical shifts. The molecular dynamics simulations indicate that the active site water molecules exchange much more frequently for WT KSI and the FFF mutant than for the Y16F and Y16S mutants. This difference is most likely due to the hydrogen-bonding interaction between Tyr57 and an active site water molecule that is persistent in the Y16F and Y16S mutants but absent in the FFF mutant and significantly less probable in WT KSI.     

Maintenance of alpha-helical structures by phenyl rings in the active-site tyrosine triad contributes to catalysis and stability of ketosteroid isomerase from Pseudomonas putida biotype B.

Ketosteroid isomerase (KSI) from Pseudomonas putida biotype B is a homodimeric enzyme catalyzing an allylic rearrangement of Delta5-3-ketosteroids at rates comparable with the diffusion-controlled limit. The tyrosine triad (Tyr14.Tyr55.Tyr30) forming a hydrogen-bond network in the apolar active site of KSI has been characterized in an effort to identify the roles of the phenyl rings in catalysis, stability, and unfolding of the enzyme. The replacement of Tyr14, a catalytic residue, with serine resulted in a 33-fold decrease of kcat, while the replacements of Tyr30 and Tyr55 with serine decreased kcat by 4- and 51-fold, respectively. The large decrease of kcat for Y55S could be due to the structural perturbation of alpha-helix A3, which results in the reorientation of the active-site residues as judged by the crystal structure of Y55S determined at 2.2 A resolution. Consistent with the analysis of the Y55S crystal structure, the far-UV circular dichroism spectra of Y14S, Y30S, and Y55S indicated that the elimination of the phenyl ring of the tyrosine reduced significantly the content of alpha-helices. Urea-induced equilibrium unfolding experiments revealed that the DeltaG(U)H2O values of Y14S, Y30S, and Y55S were significantly decreased by 11.9, 13.7, and 9.5 kcal/mol, respectively, as compared with that of the wild type. A characterization of the unfolding kinetics based on PhiU-value analysis indicates that the interactions mediated by the tyrosine triad in the native state are very resistant to unfolding. Taken together, our results demonstrate that the internal packing by the phenyl rings in the active-site tyrosine triad contributes to the conformational stability and catalytic activity of KSI by maintaining the structural integrity of the alpha-helices.     

Pseudoreversion of the catalytic activity of Y14F by the additional substitution(s) of tyrosine with phenylalanine in the hydrogen bond network of delta 5-3-ketosteroid isomerase from Pseudomonas putida biotype B

Delta5-3-ketosteroid isomerase (KSI) from Pseudomonas putida Biotype B catalyzes the allylic isomerization of Delta5-3-ketosteroids to their conjugated Delta4-isomers via a dienolate intermediate. Two electrophilic catalysts, Tyr-14 and Asp-99, are involved in a hydrogen bond network that comprises Asp-99 Odelta2...O of Wat504...Tyr-14 Oeta...Tyr-55 Oeta.Tyr-30 Oeta in the active site of P. putida KSI. Even though neither Tyr-30 nor Tyr-55 plays an essential role in catalysis by the KSI, the catalytic activity of Y14F could be increased ca. 26-51-fold by the additional Y30F and/or Y55F mutation in the hydrogen bond network. To identify the structural basis for the pseudoreversion in the KSI, crystal structures of Y14F and Y14F/Y30F/Y55F have been determined at 1.8 and 2.0 A resolution, respectively. Comparisons of the two structures near the catalytic center indicate that the hydrogen bond between Asp-99 Odelta2 and C3-O of the steroid, which is perturbed by the Y14F mutation, can be partially restored to that in the wild-type enzyme by the additional Y30F/Y55F mutations. The kinetic parameters of the tyrosine mutants with the additional D99N or D99L mutation also support the idea that Asp-99 contributes to catalysis more efficiently in Y14F/Y30F/Y55F than in Y14F. In contrast to the catalytic mechanism of Y14F, the C4 proton of the steroid substrate was found to be transferred to the C6 position in Y14F/Y30F/Y55F with little exchange of the substrate 4beta-proton with a solvent deuterium based on the reaction rate in D2O. Taken together, our findings strongly suggest that the improvement in the catalytic activity of Y14F by the additional Y30F/Y55F mutations is due to the changes in the structural integrity at the catalytic site and the resulting restoration of the proton-transfer mechanism in Y14F/Y30F/Y55F.     

Asp-99 donates a hydrogen bond not to Tyr-14 but to the steroid directly in the catalytic mechanism of Delta 5-3-ketosteroid isomerase from Pseudomonas putida biotype B

Delta 5-3-ketosteroid isomerase (KSI) catalyzes the allylic isomerization of Delta 5-3-ketosteroids at a rate approaching the diffusion limit by an intramolecular transfer of a proton. Despite the extensive studies on the catalytic mechanism, it still remains controversial whether the catalytic residue Asp-99 donates a hydrogen bond to the steroid or to Tyr-14. To clarify the role of Asp-99 in the catalysis, two single mutants of D99E and D99L and three double mutants of Y14F/D99E, Y14F/D99N, and Y14F/D99L have been prepared by site-directed mutagenesis. The D99E mutant whose side chain at position 99 is longer by an additional methylene group exhibits nearly the same kcat as the wild-type while the D99L mutant exhibits ca. 125-fold lower kcat than that of the wild-type. The mutations made at positions 14 and 99 exert synergistic or partially additive effect on kcat in the double mutants, which is inconsistent with the mechanism based on the hydrogen-bonded catalytic dyad, Asp-99 COOH...Tyr-14 OH...C3-O of the steroid. The crystal structure of D99E/D38N complexed with equilenin, an intermediate analogue, at 1.9 A resolution reveals that the distance between Tyr-14 O eta and Glu-99 O epsilon is ca. 4.2 A, which is beyond the range for a hydrogen bond, and that the distance between Glu-99 O epsilon and C3-O of the steroid is maintained to be ca. 2.4 A, short enough for a hydrogen bond to be formed. Taken together, these results strongly support the idea that Asp-99 contributes to the catalysis by donating a hydrogen bond directly to the intermediate.     

Contribution of conserved amino acids at the dimeric interface to the conformational stability and the structural integrity of the active site in ketosteroid isomerase from Pseudomonas putida biotype B

Ketosteroid isomerase (KSI) from Pseudomonas putida biotype B is a homodimeric enzyme catalyzing an allylic isomerization of Delta(5)-3-ketosteroids at a rate of the diffusion-controlled limit. The dimeric interactions mediated by Arg72, Glu118, and Asn120, which are conserved in the homologous KSIs, have been characterized in an effort to investigate the roles of the conserved interface residues in stability, function and structure of the enzyme. The interface residues were replaced with alanine to generate the interface mutants R72A, E118A, N120A and E118A/N120A. Equilibrium unfolding analysis revealed that the DeltaG(U)(H(2)O) values for the R72A, E118A, N120A, and E118A/N120A mutants were decreased by about 3.8, 3.9, 7.8, and 9.5 kcal/mol, respectively, relative to that of the wild-type enzyme. The interface mutations not only decreased the k(cat)/K(M) value by about 8- to 96-fold, but also increased the K(D) value for d-equilenin, a reaction intermediate analogue, by about 7- to 17.5-fold. The crystal structure of R72A determined at 2.5 A resolution and the fluorescence spectra of all the mutants indicated that the interface mutations altered the active-site geometry and resulted in the decreases of the conformational stability as well as the catalytic activity of KSI. Taken together, our results strongly suggest that the conserved interface residues contribute to stabilization and structural integrity of the active site in the dimeric KSI.     

A theoretical model of the catalytic mechanism of the Delta5-3-ketosteroid isomerase reaction

The present paper describes a theoretical approach to the catalytic reaction mechanism involved in the conversion of 5-androstene-3,17-dione to 4-androstene-3,17-dione. The model incorporates the side chains of the residues tyrosine (Tyr(14)), aspartate (Asp(38)) and aspartic acid (Asp(99)) of the enzyme Delta(5)-3-ketosteroid isomerase (KSI; EC 5.3.3.1). The reaction involves two steps: first, Asp(38) acts as a base, abstracting the 4beta-H atom (proton) from C-4 of the steroid to form a dienolate as the intermediate; next, the intermediate is reketonized by proton transfer to the 6beta-position. Each step goes through its own transition state. Functional groups of the Tyr(14) and Asp(99) side chains act as hydrogen bond donors to the O1 atom of the steroid, providing stability along the reaction coordinate. Calculations were assessed at high level Hartree-Fock theory, using the 6-31G(*) basis set and the most important physicochemical properties involved in each step of the reaction, such as total energy, hardness, and dipole moment. Likewise, to explain the mechanism of reaction, highest occupied molecular orbital (HOMO) and lowest unoccupied molecular orbital (LUMO), atomic orbital contributions to frontier orbitals formation, encoded electrostatic potentials, and atomic charges were used. Energy minima and transition state geometries were confirmed by vibrational frequency analysis. The mechanism described herein accounts for all of the properties, as well as the flow of atomic charges, explaining both catalytic mechanism and proficiency of KSI.     

Mutational analysis of the three cysteines and active-site aspartic acid 103 of ketosteroid isomerase from Pseudomonas putida biotype B.

In order to clarify the roles of three cysteines in ketosteroid isomerase (KSI) from Pseudomonas putida biotype B, each of the cysteine residues has been changed to a serine residue (C69S, C81S, and C97S) by site-directed mutagenesis. All cysteine mutations caused only a slight decrease in the k(cat) value, with no significant change of Km for the substrate. Even modification of the sulfhydryl group with 5,5'-dithiobis(2-nitrobenzoic acid) has almost no effect on enzyme activity. These results demonstrate that none of the cysteines in the KSI from P. putida is critical for catalytic activity, contrary to the previous identification of a cysteine in an active-site-directed photoinactivation study of KSI. Based on the three-dimensional structures of KSIs with and without dienolate intermediate analog equilenin, as determined by X-ray crystallography at high resolution, Asp-103 was found to be located within the range of the hydrogen bond to the equilenin. To assess the role of Asp-103 in catalysis, Asp-103 has been replaced with either asparagine (D103N) or alanine (D103A) by site-directed mutagenesis. For D103A mutant KSI there was a significant decrease in the k(cat) value: the k(cat) of the mutant was 85-fold lower than that of the wild-type enzyme; however, for the D103N mutant, which retained some hydrogen bonding capability, there was a minor decrease in the k(cat) value. These findings support the idea that aspartic acid 103 in the active site is an essential catalytic residue involved in catalysis by hydrogen bonding to the dienolate intermediate.     

Contribution of a Low-Barrier Hydrogen Bond to Catalysis Is Not Significant in Ketosteroid Isomerase

Low-barrier hydrogen bonds (LBHBs) have been proposed to have important influences on the enormous reaction rate increases achieved by many enzymes. Δ⁵-3-ketosteroid isomerase (KSI) catalyzes the allylic isomerization of Δ⁵-3-ketosteroid to its conjugated Δ⁴-isomers at a rate that approaches the diffusion limit. Tyr14, a catalytic residue of KSI, has been hypothesized to form an LBHB with the oxyanion of a dienolate steroid intermediate generated during the catalysis. The unusual chemical shift of a proton at 16.8 ppm in the nuclear magnetic resonance spectrum has been attributed to an LBHB between Tyr14 Oη and C3-O of equilenin, an intermediate analogue, in the active site of D38N KSI. This shift in the spectrum was not observed in Y30F/Y55F/D38N and Y30F/Y55F/Y115F/D38N mutant KSIs when each mutant was complexed with equilenin, suggesting that Tyr14 could not form LBHB with the intermediate analogue in these mutant KSIs. The crystal structure of Y30F/Y55F/Y115F/D38N-equilenin complex revealed that the distance between Tyr14 Oη and C3-O of the bound steroid was within a direct hydrogen bond. The conversion of LBHB to an ordinary hydrogen bond in the mutant KSI reduced the binding affinity for the steroid inhibitors by a factor of 8.1–11. In addition, the absence of LBHB reduced the catalytic activity by only a factor of 1.7–2. These results suggest that the amount of stabilization energy of the reaction intermediate provided by LBHB is small compared with that provided by an ordinary hydrogen bond in KSI.     

Binding of 2-naphthols to D38E mutants of 3-oxo-Delta 5-steroid isomerase: variation of ligand ionization state with the nature of the electrophilic component

3-Oxo-Delta(5)-steroid isomerase (KSI) catalyzes the isomerization of a variety of 3-oxo-Delta(5)-steroids to their conjugated Delta(4) isomers. The mechanism involves sequential enolization and ketonization, with Asp-38 acting to transfer a proton from C-4 to C-6 through a dienol(ate) intermediate. We have previously proposed that this intermediate is anionic, with stabilization provided from direct hydrogen bonding from Tyr-14 and Asp-99 to the oxygen of the steroid. In this work, we analyze the binding of substituted 2-naphthols, which are analogues of the intermediate dienol, to the D38E KSI mutant and the corresponding double mutants lacking one of the two electrophilic groups (D38E/Y14F and D38E/D99A). The binding of these naphthols to the mutant KSIs at pH 7 is described by the modified Bronsted equation: log K(D) = alpha(pK(a)) + constant, where K(D) is the dissociation constant of the complex. The high value of alpha for D38E (alpha = 0.87 +/- 0.06) indicates that the negative charge in these D38E-naphthol complexes is localized almost exclusively on the bound ligand. In contrast, values of alpha for the double mutants (alpha = 0.28 +/- 0.02 for D38E/Y14F and alpha = 0.25 +/- 0.02 for D38E/D99A) are consistent with very little negative charge on the oxygen of the bound naphthol. Ultraviolet spectra of 5-nitro-2-naphthol and the fluorescence spectra of equilenin bound to these mutants support this interpretation. Extrapolation of these results to the intermediate in the catalytic reaction suggests that for the reaction with D38E, the intermediate is a negatively charged dienolate with hydrogen bonding from both Tyr-14 and Asp-99. Removal of either one of these H-bond donors (Tyr-14 or Asp-99) causes destabilization of the anion and results in a dienol enzyme-intermediate complex rather than a dienolate.     

Structure of a conjugating enzyme-ubiquitin thiolester intermediate reveals a novel role for the ubiquitin tail

BACKGROUND: Ubiquitin-conjugating enzymes (E2s) are central enzymes involved in ubiquitin-mediated protein degradation. During this process, ubiquitin (Ub) and the E2 protein form an unstable E2-Ub thiolester intermediate prior to the transfer of ubiquitin to an E3-ligase protein and the labeling of a substrate for degradation. A series of complex interactions occur among the target substrate, ubiquitin, E2, and E3 in order to efficiently facilitate the transfer of the ubiquitin molecule. However, due to the inherent instability of the E2-Ub thiolester, the structural details of this complex intermediate are not known. RESULTS: A three-dimensional model of the E2-Ub thiolester intermediate has been determined for the catalytic domain of the E2 protein Ubc1 (Ubc1(Delta450)) and ubiquitin from S. cerevisiae. The interface of the E2-Ub intermediate was determined by kinetically monitoring thiolester formation by 1H-(15)N HSQC spectra by using combinations of 15N-labeled and unlabeled Ubc1(Delta450) and Ub proteins. By using the surface interface as a guide and the X-ray structures of Ub and the 1.9 A structure of Ubc1(Delta450) determined here, docking simulations followed by energy minimization were used to produce the first model of a E2-Ub thiolester intermediate. CONCLUSIONS: Complementary surfaces were found on the E2 and Ub proteins whereby the C terminus of Ub wraps around the E2 protein terminating in the thiolester between C88 (Ubc1(Delta450)) and G76 (Ub). The model supports in vivo and in vitro experiments of E2 derivatives carrying surface residue substitutions. Furthermore, the model provides insights into the arrangement of Ub, E2, and E3 within a ternary targeting complex.     

Substrate polarization by residues in delta 5-3-ketosteroid isomerase probed by site-directed mutagenesis and UV resonance Raman spectroscopy.

delta 5-3-Ketosteroid isomerase (KSI: EC 5.3.3.1) of Pseudomonas testosteroni catalyzes the isomerization of delta 5-3-ketosteroids to delta 4-3-ketosteroids by the stereospecific transfer of the steroid 4 beta-proton to the 6 beta-position, using Tyr-14 as a general acid and Asp-38 as a base. Ultraviolet resonance Raman (UVRR) spectra have been obtained for the catalytically active double mutant Y55F + Y88F, which retains Tyr-14 as the only tyrosine residue (referred to as the Y14(0) mutant), and the Y14F mutant, which has 50,000-fold lower activity. The UVRR results establish that binding of the product analog and competitive inhibitors 19-nortestosterone or 4-fluoro-19-nortestosterone to the Y14(0) mutant does not result in the formation of deprotonated Tyr-14. The UVRR spectra of the steroid inhibitors show large decreases in the vinyl and carbonyl stretching frequencies on binding to the Y14(0) enzyme but not on binding to the Y14F enzyme. These changes cannot be mimicked by protonation of the steroids. For 19-nortestosterone, the vinyl and carbonyl stretching frequencies shift down (with respect to the values in aqueous solution) by 18 and 27 cm-1, respectively, on binding to Y14(0) KSI. It is proposed that the changes in the steroid resonance Raman spectrum arise from polarization of the enone moiety via the close proximity of the charged Asp-38 side chain to the vinyl group and the directional hydrogen bond between Tyr-14 and the 3-carbonyl oxygen of the steroid enone. The 230-nm-excited UVRR spectra do not, however, show changes that are characteristic of strong hydrogen bonding from the tyrosine hydrogen. It is proposed that this hydrogen bonding is compensated by a second hydrogen bond to the Tyr-14 oxygen from another protein residue. UVRR spectra of the Y14(0) enzyme obtained using 200 nm excitation show enhancement of the amide II and S Raman bands. The secondary structure of KSI was estimated from the amide II and S intensities and was found to be low in alpha-helical structure. The alpha-helix content was estimated to be in the range of 0-25% (i.e., 10 +/- 15%).     

Anesthetic interaction with ketosteroid isomerase: insights from molecular dynamics simulations

The nature and the sites of interactions between anesthetic halothane and homodimeric Delta5-3-ketosteroid isomerase (KSI) are characterized by flexible ligand docking and confirmed by 1H-15N NMR. The dynamics consequence of halothane interaction and the implication of the dynamic changes to KSI function are studied by multiple 5-ns molecular dynamics simulations in the presence and absence of halothane. Both docking and MD simulations show that halothane prefer the amphiphilic dimeric interface to the hydrophobic active site of KSI. Halothane occupancy at the dimer interface disrupted the intersubunit hydrogen bonding formed either directly through side chains of polar residues or indirectly through the mediation of the interfacial water molecules. Moreover, in the presence of halothane, the exchange rate of the bound waters with bulk water was increased. Halothane perturbation to the dimer interface affected the overall flexibility of the active site. This action is likely to contribute to the halothane-induced reduction of the KSI activity. The allosteric halothane modulation of the dynamics-function relationship of KSI without direct competition at the enzymatic active sites may be generalized to offer a unifying explanation of anesthetic action on a diverse range of multidomain neuronal proteins that are potentially relevant to clinical general anesthesia.     

Second step of hydrolytic dehalogenation in haloalkane dehalogenase investigated by QM/MM methods

Mechanistic studies on the hydrolytic dehalogenation catalyzed by haloalkane dehalogenases are of importance for environmental and industrial applications. Here, Car-Parrinello (CP) and ONIOM hybrid quantum-mechanical/molecular mechanics (QM/MM) are used investigate the second reaction step of the catalytic cycle, which comprises a general base-catalyzed hydrolysis of an ester intermediate (EI) to alcohol and free enzyme. We focus on the enzyme LinB from Sphingomonas paucimobilis UT26, for which the X-ray structure at atomic resolution is available. In agreement with previous proposals, our calculations suggest that a histidine residue (His272), polarized by glutamate (Glu132), acts as a base, accepting a proton from the catalytic water molecule and transferring it to an alcoholate ion. The reaction proceeds through a metastable tetrahedral intermediate, which shows an easily reversed reaction to the EI. In the formation of the products, the protonated aspartic acid (Asp108) can easily adopt conformation of the relaxed state found in the free enzyme. The overall free energy barrier of the reaction calculated by potential of the mean force integration using CP-QM/MM calculations is equal to 19.5 +/- 2 kcal . mol(-1). The lowering of the energy barrier of catalyzed reaction with respect to the water reaction is caused by strong stabilization of the reaction intermediate and transition state and their preorganization by electrostatic field of the enzyme.     

Energetics of 3-oxo-delta 5-steroid isomerase: source of the catalytic power of the enzyme

Knowledge of the partitioning of the putative dienol intermediate (2) by steroid isomerase (KSI) (Hawkinson et al. 1991), in conjunction with various steady-state kinetic parameters, allows elucidation of the detailed free energy profile for the KSI-catalyzed conversion of 5-androstene-3,17-dione (1) to 4-androstene-3,17-dione (3). This free energy profile shows four kinetically significant energy barriers (substrate binding, the two chemical steps, and dissociation of product) that must be traversed upon conversion of 1 to 3. Thus, no single step of the catalytic cycle is cleanly rate-limiting. The source of the catalytic power of KSI is discussed via comparison of the free energy profile for the KSI-catalyzed isomerization with those for the acetate-catalyzed isomerization and the aqueous reaction at pH 7. Similarities between the energetics of the KSI-catalyzed and triosephosphate isomerase catalyzed reactions are also noted.     

Structure-based reconstruction of a Mycobacterium hypothetical protein into an active Δ5–3-ketosteroid isomerase

Protein engineering based on structure homology holds the potential to engineer steroid-transforming enzymes on demand. Based on the genome sequencing analysis of industrial Mycobacterium strain HGMS2 to produce 4-androstene-3,17-dione (4-AD), three hypothetical proteins were predicted as putative Δ⁵–3-ketosteroid isomerases (KSIs) to catalyze an intramolecular proton transfer involving the transformation of 5-androstene-3,17-dione (5-AD) into 4-AD, which were defined as mKSI228, mKSI291 and mKSI753. Activity assays indicated that mKSI228 and mKSI291 exhibited weak activity, as low as 0.7% and 1.5%, respectively, of a well-studied and highly active KSI from Pseudomonas putida KSI (pKSI), while mKSI753 had no activity similar to Mycobacterium tuberculosis KSI (mtKSI). Although the 3D structures of the putative mKSIs were homologous to pKSI, their amino acid sequences were significantly different from those of pKSI and tKSI. Thus, by use of these two KSIs as homology models, we were able to convert the low-active mKSI291 into a high-active active KSI by site-directed mutagenesis. On the other hand, an X-ray crystallographic structure of mKSI291 identified a water molecule in its active site. This unique water molecule might function as a bridge to connect Ser-OH, Tyr57-OH and C3O of the intermediate form a hydrogen-bonding network that was responsible for its weak activity, compared with that of mtKSI. Our results not only demonstrated the use of a protein engineering approach to understanding KSI catalytic mechanism, but also provided an example for engineering the catalytic active sites and gaining a functional enzyme based on homologous structures.     

Acetylcholinesterase: mechanisms of covalent inhibition of H447I mutant determined by computational analyses

The reaction mechanisms of two inhibitor TFK(+) and TFK(0) binding to H447I mutant mouse acetylcholinesterase (mAChE) have been investigated by using a combined ab initio quantum mechanical/molecular mechanical (QM/MM) approach and classical molecular dynamics (MD) simulations. TFK(+) binding to the H447I mutant may proceed with a different reaction mechanism from the wild-type. A water molecule takes over the role of His447 and participates in the bond breaking and forming as a "charge relayer". Unlike in the wild-type mAChE case, Glu334, a conserved residue from the catalytic triad, acts as a catalytic base in the reaction. The calculated energy barrier for this reaction is about 8kcal/mol. These predictions await experimental verification. In the case of the neutral ligand TFK(0), however, multiple MD simulations on the TFK(0)/H447I complex reveal that none of the water molecules can be retained in the active site as a "catalytic" water. Taken together our computational studies confirm that TFK(0) is almost inactive in the H447I mutant, and also provide detailed mechanistic insights into the experimental observations.     

Oxygen exchange between acetate and the catalytic glutamate residue in glutaconate CoA-transferase from Acidaminococcus fermentans. Implications for the mechanism of CoA-ester hydrolysis.

The exchange of oxygen atoms between acetate, glutaryl-CoA, and the catalytic glutamate residue in glutaconate CoA-transferase from Acidaminococcus fermentans was analyzed using [(18)O(2)]acetate together with matrix-assisted laser desorption/ionization time of flight mass spectrometry of an appropriate undecapeptide. The exchange reaction was shown to be site-specific, reversible, and required both glutaryl-CoA and [(18)O(2)]acetate. The observed exchange is in agreement with the formation of a mixed anhydride intermediate between the enzyme and acetate. In contrast, with a mutant enzyme, which was converted to a thiol ester hydrolyase by replacement of the catalytic glutamate residue by aspartate, no (18)O uptake from H(2)(18)O into the carboxylate was detectable. This result is in accord with a mechanism in which the carboxylate of aspartate acts as a general base in activating a water molecule for hydrolysis of the thiol ester intermediate. This mechanism is further supported by the finding of a significant hydrolyase activity of the wild-type enzyme using acetyl-CoA as substrate, whereas glutaryl-CoA is not hydrolyzed. The small acetate molecule in the substrate binding pocket may activate a water molecule for hydrolysis of the nearby enzyme-CoA thiol ester.     

A tale of two isomerases: compact versus extended active sites in ketosteroid isomerase and phosphoglucose isomerase

Understanding the catalytic efficiency and specificity of enzymes is a fundamental question of major practical and conceptual importance in biochemistry. Although progress in biochemical and structural studies has enriched our knowledge of enzymes, the role in enzyme catalysis of residues that are not nearest neighbors of the reacting substrate molecule is largely unexplored experimentally. Here computational active site predictors, THEMATICS and POOL, were employed to identify functionally important residues that are not in direct contact with the reacting substrate molecule. These predictions then guided experiments to explore the active sites of two isomerases, Pseudomonas putida ketosteroid isomerase (KSI) and human phosphoglucose isomerase (PGI), as prototypes for very different types of predicted active sites. Both KSI and PGI are members of EC 5.3 and catalyze similar reactions, but they represent significantly different degrees of remote residue participation, as predicted by THEMATICS and POOL. For KSI, a compact active site of mostly first-shell residues is predicted, but for PGI, an extended active site in which residues in the first, second, and third layers around the reacting substrate are predicted. Predicted residues that have not been previously tested experimentally were investigated by site-directed mutagenesis and kinetic analysis. In human PGI, single-point mutations of the predicted second- and third-shell residues K362, H100, E495, D511, H396, and Q388 show significant decreases in catalytic activity relative to that of the wild type. The results of these experiments demonstrate that, as predicted, remote residues are very important in PGI catalysis but make only small contributions to catalysis in KSI.     

Interaction of nucleic acid bases with water molecules and formation of mismatched nucleotide pairs

The possibility of the inclusion of water molecules in the formation of mismatched nucleotide pairs was considered in relation to the mechanisms of point errors in template directed biosynthesis. Calculations of the intermolecular interaction energy for systems containing two bases and one water molecule were carried out by the method of atom-atom potential functions. There exist energy minima for each base pair, corresponding to a single N--H...O or N--H...N H-bond between the bases and H-bonding of the water molecule with both bases. The relative positions of glycosyl bonds in some of these minima are closer to those for Watson--Crick pairs, than the positions of minima for these pairs without water. For other minima, the H-bond formation between the water molecule and the two bases additionally stabilizes the relative base position in wobble-pairs with two H-bonds between the bases. The base and water positions in energy minima are compared with the positions in some pairs proposed on the basis of NMR and X-ray data for double helical oligonucleotides.     

Crystallographic evidence for active-site dynamics in the hydrolytic aldehyde dehydrogenases. Implications for the deacylation step of the catalyzed reaction

The overall chemical mechanism of the reaction catalyzed by the hydrolytic aldehyde dehydrogenases (ALDHs) involves three main steps: (1) nucleophilic attack of the thiol group of the catalytic cysteine on the carbonyl carbon of the aldehyde substrate; (2) hydride transfer from the tetrahedral thiohemiacetal intermediate to the pyridine ring of NAD(P)(+); and (3) hydrolysis of the resulting thioester intermediate (deacylation). Crystal structures of different ALDHs from several organisms-determined in the absence and presence of bound NAD(P)(+), NAD(P)H, aldehydes, or acid products-showed specific details at the atomic level about the catalytic residues involved in each of the catalytic steps. These structures also showed the conformational flexibility of the nicotinamide half of the cofactor, and of the catalytic cysteinyl and glutamyl residues, the latter being the general base that activates the hydrolytic water molecule in the deacylation step. The architecture of the ALDH active site allows for this conformational flexibility, which, undoubtedly, is crucial for catalysis in these enzymes. Focusing in the deacylation step of the ALDH-catalyzed reaction, here we review and systematize the crystallographic evidence of the structural features responsible for the conformational flexibility of the catalytic glutamyl residue, and for the positioning of the hydrolytic water molecule inside the ALDH active site. Based on the analysis of the available crystallographic data and of energy-minimized models of the thioester reaction intermediate, as well as on the results of theoretical calculations of the pK(a) of the carboxyl group of the catalytic glutamic acid in its three different conformations, we discuss the role that the conformational flexibility of this residue plays in the activation of the hydrolytic water. We also propose a critical participation in the water activation process of the peptide bond to which the catalytic glutamic acid in the intermediate conformation is hydrogen bonded.