Catalytic Diad Research Articles

Mechanistic Studies of Ubiquitin C-Terminal Hydrolase L1

Ubiquitin C-terminal hydrolases (UCHs) cleave Ub-X bonds (Ub is ubiquitin and X an alcohol, an amine, or a protein) through a thioester intermediate that is produced by nucleophilic attack of the Cys residue of a Cys-SH/His-Im catalytic diad. We are studying the mechanism of UCH-L1, a UCH that is implicated in Parkinson's disease, and now wish to report our initial findings. (i) Pre-steady-state kinetic studies for UCH-L1-catalyzed hydrolysis of Ub-AMC (AMC, 7-amido-4-methylcoumarin) indicate that k(cat) is rate-limited by acyl-enzyme formation. Thus, K(m) = K(s), the dissociation constant for the Michaelis complex, and k(cat) = k(2), the rate constant for acyl-enzyme formation. (ii) For K(assoc) (=K(s)(-)(1)), DeltaC(p) = -0.8 kcal mol(-)(1) deg(-)(1) and is consistent with coupling between substrate association and a conformational change of the enzyme. For k(2), DeltaS(++) = 0 and suggests that in the E-S, substrate and active site residues are precisely aligned for reaction. (iii) Solvent isotope effects are (D)K(assoc) = 0.5 and (D)k(2) = 0.9, suggesting that the substrate binds to a form of free enzyme in which the active site Cys exists as the thiol. In the resultant Michaelis complex, the diad has tautomerized to ion pair Cys-S(-)/His-ImH(+). Subsequent attack of thiolate produces the acyl-enzyme species. In contrast, isotope effects for association of UCH-L1 with transition-state analogue ubiquitin aldehyde suggest that an alternative mechanistic pathway can sometimes be available to UCH-L1 involving general base-catalyzed attack of Cys-SH by His-Im.

The demise of the marine cyanobacterium, Trichodesmium spp., via an autocatalyzed cell death pathway

We present experimental laboratory evidence and field observations of an autocatalyzed, programmed cell death (PCD) pathway in the nitrogen‐fixing cyanobacterium Trichodesmium spp., which forms massive blooms in the subtropical and tropical oceans. The PCD pathway was induced in response to phosphorus and iron starvation as well as high irradiance and oxidative stress. Transmission electron microscopy revealed morphological degradation of internal components including thylakoids, carboxysomes, and gas vesicles, whereas the plasma membranes remained intact. Physiologically stressed cells displayed significantly elevated endonuclease activity and terminal d‐UTP nick‐end labeling. Nucleic acid degradation was concordant with increased immunoreactivity to human caspase‐3 polyclonal antisera and enhanced cleavage of a caspase‐specific substrate, DEVD. Caspase activity was positively correlated with mortality and was inhibited by the irreversible caspase inhibitor Z‐VAD‐FMK. A search of the Trichodesmium erythraeum genome identified several protein sequences containing a conserved caspase domain structure, including the histidine‐ and cysteine‐containing catalytic diad found in true caspases, paracaspases, and metacaspases. Induction of PCD by caspase‐like proteases in a bacterial photoautotroph with an ancient evolutionary history requires a reassessment about the origins and roles of cell death cascades. This process is a previously unappriciated mortality mechanism that can lead to the termination of natural Trichodesmium blooms and that can influence the fluxes of organic matter in the ocean.

Cloning and Heterologous Expression of a Glucodextranase Gene from Arthrobacter globiformis I42, and Experimental Evidence for the Catalytic Diad of the Recombinant Enzyme

Cloning and Heterologous Expression of a Glucodextranase Gene from Arthrobacter globiformis I42, and Experimental Evidence for the Catalytic Diad of the Recombinant Enzyme

Computational study of ketosteroid isomerase: insights from molecular dynamics simulation of enzyme bound substrate and intermediate.

Delta(5)-3-Ketosteroid Isomerase (KSI) catalyzes the isomerization of 5,6-unsaturated ketosteroids to their 4,5-unsaturated isomers at a rate approaching the diffusion limit. The isomerization reaction follows a two-step general acid-base mechanism starting with Asp38-CO(2)(-) mediated proton abstraction from a sp(3)-hybridized carbon atom, alpha to carbonyl group, providing a dienolate intermediate. In the second step, Asp38-CO(2)H protonates the C6 of the intermediate providing a 4,5-unsaturated ketosteroid. The details of the mechanism have been highly controversial despite several experimental and computational studies of this enzyme. The general acid-base catalysis has been proposed to involve either a catalytic diad or a cooperative hydrogen bond mechanism. In this paper, we report our results from the 1.5 nanosecond molecular dynamics (MD) simulation of enzyme bound natural substrate (E.S) and enzyme bound intermediate (E. In) solvated in a TIP3P water box. The final coordinates from our MD simulation strongly support the cooperative hydrogen bond mechanism. The MD simulation of E.S and E. In shows that both Tyr14 and Asp99 are hydrogen bonded to the O3 of the substrate or intermediate. The average hydrogen bonding distance between Tyr14-OH and O3 becomes shorter and exhibits less fluctuation on E.S --> E. In. We also observe dynamic motions of water moving in and out of the active site in the E.S structures. This free movement of water disappears in the E. In structures. The active site is shielded by hydrophobic residues, which come together and squeeze out the waters from the active site in the E. In complex.

General base catalysis in the urate oxidase reaction: evidence for a novel Thr-Lys catalytic diad.

Urate oxidase catalyzes the oxidation of urate without the involvement of any cofactors. The gene encoding urate oxidase from Bacillus subtilis has been cloned and expressed, and the enzyme was purified and characterized. Formation of the urate dianion is believed to be a key step in the oxidative reaction. Rapid-mixing chemical quench studies provide evidence that the dianion is indeed an intermediate; at 15 degrees C the dianion forms within the mixing time of the rapid-quench instrument, and it disappears with a rate constant of 8 s(-)(1). Steady-state kinetic studies indicate that an ionizable group on the enzyme with a pK of 6.4 must be unprotonated for catalysis, and it is presumed that the role of this group is to abstract a proton from the substrate. Surprisingly, examination of the active site provided by the previously reported crystal structure does not reveal any obvious candidates to act as the general base. However, Thr 69 is hydrogen-bonded to the ligand at the active site, and Lys 9, which does not contact the ligand, is hydrogen-bonded to Thr 69. The T69A mutant enzyme has a V(max) that is 3% of wild type, and the K9M mutant enzyme has a V(max) that is 0.4% of wild type. The ionization at pH 6.4 that is observed with wild-type enzyme is absent in both of these mutants. It is proposed that these residues form a catalytic diad in which K9 deprotonates T69 to allow it to abstract the proton from the N9 position of the substrate to generate the dianion.

Molecular dynamics and quantum chemical studies on the catalytic mechanism of Delta5-3-ketosteroid isomerase: the catalytic diad versus the cooperative hydrogen bond mechanism.

To further understand Delta(5)-3-ketosteroid isomerase (KSI) catalysis, we carried out molecular dynamics (MD) simulations of the KSI dimer ligated with a substrate and reaction intermediate analogue and high level ab initio calculations on relevant enzymatic reaction models. Simulation of the enzyme-substrate complex dimer systems showed asymmetric dynamics between the two monomers, in which the hydrogen bond pattern between the substrate and active site residues in the first and the second subunits supported the cooperative hydrogen bond (CH) and the catalytic diad (CD) mechanisms, respectively. On the other hand, only the CH mechanism was supported in the MD simulation of the enzyme-intermediate complex dimer. From MP2/6-31+G**//RHF/6-31G** calculations, we found the kinetic barriers for the two reaction mechanisms were similar. The CH route afforded a greater stabilization to the enolate intermediate than did the CD counterpart. Thus, the present computational studies indicate that the CH mechanism would be favored over the CD one in the catalytic action of KSI. However, the latter could not be ruled out conclusively because of the explicit appearance of a CD configuration in the MD trajectories of the enzyme-substrate complex and because of the similar intrinsic activation barrier for the CH and CD mechanisms. The appearance of configurations that favor the CD pathway is rationalized in terms of a model in which the KSI-substrate complex does not have a strong preference for one hydrogen bonding pattern over another, while the KSI-intermediate complex favors a cooperative hydrogen bond pattern in order to stabilize the reaction intermediate. This hypothesis is supported by the ab initio calculations which indicate that the CH intermediate is more stable than the CD one by approximately 6.3 kcal/mol.

Enhanced synthesis of poly(3-hydroxybutyrate) in recombinant Escherichia coli by means of error-prone PCR mutagenesis, saturation mutagenesis, and in vitro recombination of the type II polyhydroxyalkanoate synthase gene.

Type II synthase (PhaC1(Ps)) for polyhydroxyalkanoate (PHA) from Pseudomonas sp. 61-3 was subjected to an in vitro evolution system including PCR-mediated mutagenesis in order to improve the function of PhaC1(Ps) in terms of its ability to produce poly(3-hydroxybutyrate) [P(3HB)] in recombinant Escherichia coli. Based on our established in vivo assay system, two positions (Ser325 and Gln481) where mutations provided remarkable increases in P(3HB) synthesis were identified. Saturation mutagenesis at these positions was carried out to explore whether there might be more beneficial sequences for P(3HB) synthesis than those identified in the point mutation library. As a result, five single mutants [S325C (T) and Q481M (K, R)] gave rise to highly enhanced P(3HB) synthesis. Drastically enhanced P(3HB) synthesis (up to 340- to 400-fold the amount of that of the wild type) was further achieved by generation of all five variants of the double mutants combining the codons for residues 325/481. It is feasible that the replacement of Ser (specific for type II synthase) by Thr (specific for type I synthase) at position 325 resulted in acquiring greater P(3HB) synthesis ability as exhibited by type I synthases. The other hot spot, 481, that positively contributes to enhanced P(3HB) synthesis is located adjacent to a His479, a residue that forms a putative catalytic diad that can be inferred by sequence alignment.

Dynamics of RnaseSa: A Simulation Perspective Complementary to NMR/X-ray

Molecular dynamics simulations of Ribonuclease Sa yield properties that are compared to data extracted from crystallography and NMR. Similar distributions of static/average as well as dynamic properties are found. The pattern of fluctuations in the backbone torsions as revealed by order parameters is conserved between different techniques. The character of concerted displacements between the different techniques is qualitatively similar. The extent of variation of conformation during the simulations is much higher than is observed by any other technique and is confined to a subspace that differs from that describing the major variation in the observed structures. The functional motions deduced are composed of a highly mobile His85 coupled to a less mobile Glu54 as forming the catalytic diad, in concert with the motion of loops lining the active site. Two of these loops have been found to exist in three distinct conformational substates: the C-terminal part of the α−β2 loop from NMR and the β2−β3 loop in the simulations. The simulations present a view of the dynamics of Rnase Sa that is different but complementary to NMR and X-ray, and together the data reveal a more comprehensive picture of the protein's “real” dynamics than from any method alone.

A catalytic diad involved in substrate-assisted catalysis: NMR study of hydrogen bonding and dynamics at the active site of phosphatidylinositol-specific phospholipase C.

Phosphatidylinositol-specific phospholipase Cs (PI-PLCs, EC 3.1.4.10) are ubiquitous enzymes that cleave phosphatidylinositol or phosphorylated derivatives, generating second messengers in eukaryotic cells. A catalytic diad at the active site of Bacillus cereus PI-PLC composed of aspartate-274 and histidine-32 was postulated from the crystal structure to form a catalytic triad with the 2-OH group of the substrate [Heinz, D. W., et al. (1995) EMBO J. 14, 3855-3863]. This catalytic diad has been observed directly by proton NMR. The single low-field line in the 1H NMR spectrum is assigned by site-directed mutagenesis: The peak is present in the wild type but absent in the mutants H32A and D274A, and arises from the histidine Hdelta1 forming the Asp274-His32 hydrogen bond. This hydrogen is solvent-accessible, and exchanges slowly with H2O on the NMR time scale. The position of the low-field peak shifts from 16.3 to 13.8 ppm as the pH is varied from 4 to 9, reflecting a pKa of 8.0 at 6 degrees C, which is identified with the pKa of His32. The Hdelta1 signal is modulated by rapid exchange of the Hepsilon2 with the solvent. Estimates of the exchange rate as a function of pH and protection factors are derived from a line shape analysis. The NMR behavior is remarkably similar to that of the serine proteases. The postulated function of the Asp274-His32 diad is to hydrogen-bond with the 2-OH of phosphatidylinositol (PI) substrate to form a catalytic triad analogous to Asp-His-Ser of serine proteases. This is an example of substrate-assisted catalysis where the substrate provides the catalytic nucleophile of the triad. This hydrogen bond becomes shorter as the imidazole is protonated, suggesting it is stronger in the transition state, contributing further to the catalytic efficiency. The hydrogen bond fits the NMR criteria for a short, strong hydrogen bond, i.e., a highly deshielded proton resonance, bond length of 2.64 +/- 0.04 A at pH 6 measured by NMR, a D/H fractionation factor significantly lower than 1.0, and a protection factor > or = 100.

The dihydroxyacetone kinase of Escherichia coli utilizes a phosphoprotein instead of ATP as phosphoryl donor

The dihydroxyacetone kinase (DhaK) of Escherichia coli consists of three soluble protein subunits. DhaK (YcgT; 39.5 kDa) and DhaL (YcgS; 22.6 kDa) are similar to the N- and C-terminal halves of the ATP-dependent DhaK ubiquitous in bacteria, animals and plants. The homodimeric DhaM (YcgC; 51.6 kDa) consists of three domains. The N-terminal dimerization domain has the same fold as the IIA domain (PDB code 1PDO) of the mannose transporter of the bacterial phosphoenolpyruvate:sugar phosphotransferase system (PTS). The middle domain is similar to HPr and the C-terminus is similar to the N-terminal domain of enzyme I (EI) of the PTS. DhaM is phosphorylated three times by phosphoenolpyruvate in an EI- and HPr-dependent reaction. DhaK and DhaL are not phosphorylated. The IIA domain of DhaM, instead of ATP, is the phosphoryl donor to dihydroxyacetone (Dha). Unlike the carbohydrate-specific transporters of the PTS, DhaK, DhaL and DhaM have no transport activity.

Involvement of the Arg−Asp−His Catalytic Triad in Enzymatic Cleavage of the Phosphodiester Bond

Phosphatidylinositol-specific phospholipase C (PI-PLC) catalyzes the cleavage of the P-O bond in phosphatidylinositol via intramolecular nucleophilic attack of the 2-hydroxyl group of inositol on the phosphorus atom. Our earlier stereochemical and site-directed mutagenesis studies indicated that this reaction proceeds by a mechanism similar to that of RNase A, and that the catalytic site of PI-PLC consists of three major components analogous to those observed in RNase A, the His32 general base, the His82 general acid, and Arg69 acting as a phosphate-activating residue. In addition, His32 is associated with Asp274 in forming a catalytic triad with inositol 2-hydroxyl, and His82 is associated with Asp33 in forming a catalytic diad. The focus of this work is to provide a global view of the mechanism, assess cooperation between various catalytic residues, and determine the origin of enzyme activation by the hydrophobic leaving group. To this end, we have investigated kinetic properties of Arg69, Asp33, and His82 mutants with phosphorothioate substrate analogues which feature leaving groups of varying hydrophobicity and pK(a). Our results indicate that interaction of the nonbridging pro-S oxygen atom of the phosphate group with Arg69 is strongly affected by Asp33, and to a smaller extent by His82. This result in conjunction with those obtained earlier can be rationalized in terms of a novel, dual-function triad comprised of Arg69, Asp33, and His82 residues. The function of this triad is to both activate the phosphate group toward the nucleophilic attack and to protonate the leaving group. In addition, Asp33 and His82 mutants displayed much smaller degrees of activation by the fatty acid-containing leaving group as compared to the wild-type (WT) enzyme, and the level of activation was significantly reduced for substrates featuring the leaving group with low pK(a) values. These results strongly suggest that the assembly of the above three residues into the fully catalytically competent triad is controlled by the hydrophobic interactions of the enzyme with the substrate leaving group.

Identification of essential active-site residues in the cyanogenic β-glucosidase (linamarase) from cassava (Manihot esculenta Crantz) by site-directed mutagenesis

The coding sequence of the mature cyanogenic β-glucosidase (β-glucoside glucohydrolase, EC 3.2.1.21; linamarase) was cloned into the vector pYX243 modified to contain the SUC2 yeast secretion signal sequence and expressed in Saccharomyces cerevisiae. The recombinant enzyme is active, glycosylated and showed similar stability to the plant protein. Michaelis constants for hydrolysis of the natural substrate, linamarin (Km = 1.06mM) and the synthetic p-nitrophenyl β-D-glucopyranoside (PNP-Glc; Km = 0.36mM), as well as apparent pKa values of the free enzyme and the enzymeŐsubstrate complexes (pKE1 = 4.4Ő4.8, pKE2 = 6.7Ő7.2, pKES1 = 3.9Ő4.4, pKES2 = 8.3) were very similar to those of the plant enzyme. Site-directed mutagenesis was carried out to study the function of active-site residues based on a homology model generated for the enzyme using the MODELLER program. Changing Glu-413 to Gly destroyed enzyme activity, consistent with it being the catalytic nucleophile. The Gln-339Glu mutation also abolished activity, confirming a function in positioning the catalytic diad. The Ala-201Val mutation shifted the pKa of the acid/base catalyst Glu-198 from 7.22 to 7.44, reflecting a change in its hydrophobic environment. A Phe-269Asn change increased Km for linamarin hydrolysis 16-fold (16.1mM) and that for PNP-Glc only 2.5-fold (0.84mM), demonstrating that Phe-269 contributes to the cyanogenic specificity of the cassava β-glucosidase.

Solution structure of Delta5-3-ketosteroid isomerase complexed with the steroid 19-nortestosterone hemisuccinate

The solution structure of the ketosteroid isomerase homodimer complexed with the product analogue 19-nortestosterone hemisuccinate (19-NTHS) was solved by heteronuclear multidimensional NMR methods using 1647 distance restraints, 77 dihedral angle (phi) restraints, and 67 hydrogen bond restraints per monomer. The refined secondary structure of each subunit consists of three alpha-helices, eight beta-strands, four turns, and two beta-bulges. The beta-strands form a mixed beta-sheet. One of the five proline residues, Pro-39, is cis and begins a nonclassical turn. A self-consistent ensemble of 15 tertiary/quaternary structures of the enzyme dimer-steroid complex, with no distance violations greater than 0.35 A, was generated by simulated annealing and energy minimization with the program X-PLOR. The mean pairwise RMSD of the secondary structural elements was 0.63 A for the average subunit and 1.25 A for the dimer. Within each subunit, the three alpha-helices are packed onto the concave surface of the beta-sheet with a groove between them into which the steroid binds at a site defined by 14 intermolecular distances. In the productive complex, Tyr-14, from alpha-helix 1, approaches both Asp-99 and the 3-keto group of 19-NTHS while, from beta-strand 1, the carboxylate of Asp-38 approaches the beta-face of the steroid near C4 and C6, between which it transfers a proton during catalysis. Thus the solution structure of the isomerase-steroid complex can accommodate the catalytic diad mechanism in which Asp-99 donates a hydrogen bond to Tyr-14 which in turn is hydrogen bonded to the 3-oxygen of the steroid. While direct hydrogen bonding of Asp-99 to the steroid oxygen is less likely, it cannot be excluded. All other interactions of the steroid with the enzyme are hydrophobic. The dimer interface, which is between the convex surfaces of the beta-sheets, is defined by 28 intersubunit NOEs between hydrophobic residues in the 13C-filtered NOESY-HSQC spectrum of a 13C/12C-heterolabeled dimer. Both hydrophobic and polar interactions occur at the dimer interface which contains no space that would permit additional steroid binding. Comparison of the complexed enzyme with the solution structure of the free enzyme [Wu et al. (1997) Science 276, 415-418] reveals that the three helices change position in the steroid complex, becoming more closely packed onto the concave surface of the beta-sheet, thus bringing Tyr-14 closer to Asp-99 and the substrate. Comparison of the enzyme-steroid complex in solution with the free enzyme in the crystalline state reveals similar differences between the positions of the helices.

X-ray studies on two forms of bovine β-trypsin crystals in neat cyclohexane

Two orthorhombic forms ( V m values are 2.3 and 3.0 Å 3/Da) of bovine β-trypsin crystals in neat cyclohexane were determined to 1.93 Å resolution, by X-ray diffraction. Both structures in organic solvent are similar to those in aqueous solution. In the high packing density form, one cyclohexane molecule is found in a hydrophobic site near the active center. One sulfate locates at the active site with hydrogen or salt bond to the Ser-His catalytic diad, and five more sulfates bind on the molecular surface. The conformation of the side chains near the sulfates changed greatly. In the low packing density form, one cyclohexane and three sulfates are found. In both structures, one benzamidine molecule locates at the hydrophobic pocket of the active center. Most water molecules on the enzyme surface are retained except some with high temperature factors.

Solution Structure of Δ5-3-Ketosteroid Isomerase Complexed with the Steroid 19-Nortestosterone Hemisuccinate,

The solution structure of the ketosteroid isomerase homodimer complexed with the product analogue 19-nortestosterone hemisuccinate (19-NTHS) was solved by heteronuclear multidimensional NMR methods using 1647 distance restraints, 77 dihedral angle (phi) restraints, and 67 hydrogen bond restraints per monomer. The refined secondary structure of each subunit consists of three alpha-helices, eight beta-strands, four turns, and two beta-bulges. The beta-strands form a mixed beta-sheet. One of the five proline residues, Pro-39, is cis and begins a nonclassical turn. A self-consistent ensemble of 15 tertiary/quaternary structures of the enzyme dimer-steroid complex, with no distance violations greater than 0.35 A, was generated by simulated annealing and energy minimization with the program X-PLOR. The mean pairwise RMSD of the secondary structural elements was 0.63 A for the average subunit and 1.25 A for the dimer. Within each subunit, the three alpha-helices are packed onto the concave surface of the beta-sheet with a groove between them into which the steroid binds at a site defined by 14 intermolecular distances. In the productive complex, Tyr-14, from alpha-helix 1, approaches both Asp-99 and the 3-keto group of 19-NTHS while, from beta-strand 1, the carboxylate of Asp-38 approaches the beta-face of the steroid near C4 and C6, between which it transfers a proton during catalysis. Thus the solution structure of the isomerase-steroid complex can accommodate the catalytic diad mechanism in which Asp-99 donates a hydrogen bond to Tyr-14 which in turn is hydrogen bonded to the 3-oxygen of the steroid. While direct hydrogen bonding of Asp-99 to the steroid oxygen is less likely, it cannot be excluded. All other interactions of the steroid with the enzyme are hydrophobic. The dimer interface, which is between the convex surfaces of the beta-sheets, is defined by 28 intersubunit NOEs between hydrophobic residues in the 13C-filtered NOESY-HSQC spectrum of a 13C/12C-heterolabeled dimer. Both hydrophobic and polar interactions occur at the dimer interface which contains no space that would permit additional steroid binding. Comparison of the complexed enzyme with the solution structure of the free enzyme [Wu et al. (1997) Science 276, 415-418] reveals that the three helices change position in the steroid complex, becoming more closely packed onto the concave surface of the beta-sheet, thus bringing Tyr-14 closer to Asp-99 and the substrate. Comparison of the enzyme-steroid complex in solution with the free enzyme in the crystalline state reveals similar differences between the positions of the helices.

Hydrogen bonding at the active site of delta 5-3-ketosteroid isomerase.

The solution secondary structure of the highly active Y55F/Y88F "Tyr-14-only" mutant of delta 5-3-ketosteroid isomerase complexed with 19-nortestosterone hemisuccinate has been shown to consist of three helices, a six-stranded mixed beta-sheet, and five turns. The steroid binds near the general acid, Tyr-14, on helix 1, near the general base, Asp-38, on the first strand of the beta-sheet, and on the hydrophobic face of the beta-sheet [Zhao, Q., Abeygunawardana, C., & Mildvan, A. S. (1997) Biochemistry 36, 3458-3472]. On this hydrophobic face, Asp-99 is the only polar residue. Free isomerase shows a deshielded exchangeable proton resonance at 13.1 ppm assigned to the N epsilon H of neutral His-100. Its fractionation factor (phi = 0.79) and slow exchange with solvent suggest it to be buried or involved in an H-bond. The binding of dihydroequilenin or estradiol to isomerase induces the appearance of two additional deshielded proton resonances, one at 18.2 ppm assigned to the gamma-carboxyl proton of Asp-99, and the other, at 11.6 ppm, assigned to the zeta-OH proton of Tyr-14. While mutation of Asp-99 to Ala results in the disappearance of only the resonance near 18 ppm [Wu, R. W., Ebrahemian, S., Zwrotny, M. E., Thornberg, L. D., Perez-Alverado, G. C., Brothers, P., Pollack, R. M., & Summers, M. F. (1997) Science 276, 415-418], both of these resonances disappear in mutants lacking Tyr-14, suggesting an H-bonded catalytic diad, Asp-99-COOH--Tyr14-OH--O-steroid enolate. The catalytic diad is further supported by NOEs from the beta 1 and beta 2 protons of Asp-99 to the epsilon protons of Tyr-14, and from the zeta-OH proton of Tyr-14 to the gamma-carboxyl proton of Asp-99, indicating close proximity of these two residues, and by other data from the literature. A strong, low-barrier H-bond between Asp-99 and Tyr-14 is indicated by the 6.2 ppm deshielding, low fractionation factor (phi = 0.34) and slow exchange of the resonance at 18.2 ppm. A normal H-bond between Tyr-14 and the steroid is indicated by the 1.8 ppm deshielding, fractionation factor of 0.97 and the slow exchange of the resonance at 11.6 ppm. It is suggested that the 10(4.7)-fold contribution of Tyr-14 to catalysis is made possible by strong H-bonding from Asp-99 in the catalytic diad which strengthens general acid catalysis by Tyr-14. It is also noted that highly deshielded proton resonance on enzymes between 15 and 20 ppm, assigned to low-barrier H-bonds, generally involve carboxyl groups.

Mechanism of action of aspartic proteinases: application of transition-state analogue theory.

Applying the semiempirical MO methods AM1 and PM3 as well as the density functional theory to the model of the catalytic site composed of ca. 160-190 atoms, we have carried out studies aimed at the explanation of three aspects of the mechanism of action of aspartic proteinases: the site of dissociation within the catalytic diad COOH/COO- (i) in the free enzyme and (ii) in the Michaelis complex, and (iii) the energy changes associated with the catalytic paths. We have found that the state of dissociation within the catalytic diad is ligand-sensitive. In the free enzyme and in the intermediate complexes, Asp33 prefers to be dissociated with the outer oxygen of Asp213 protonated, while in the Michaelis and product complexes the opposite holds true. This is in agreement with recent mechanistic hypotheses and with some experimental results by FTIR and NMR. The energy diagram for the catalysis indicates that electronic effects are responsible most of all for the relative reduction of energy of the intermediates and possibly transition states on the catalytic reaction path. The shape of the diagram qualitatively agrees with the transition-state analogue theory for the enzymatic reactions.

Phospholipase A2 engineering. 10. The aspartate...histidine catalytic diad also plays an important structural role

The «catalytic triad» of serine proteases and lipases and the «catalytic diad» of phospholipase A 2 (PLA2) are the key catalytic machineries of these enzymes. We present evidence that the catalytic diad of PLA2 also plays an important structural role. The His-48 of PLA2 was changed to Asn, Gln, and Ala; the resulting mutants are named H48N, H48Q, and H48A, respectively. The amide nitrogen atoms of Asn and Gln mimic the δ 1 -nitrogen and the e 2 -nitrogen, respectively, of His. According to the crystal structure, the δ 1 -nitrogen is the actual general base while the e 2 -nitrogen is hydrogen-bonded to Asp-99

Crystal structure of the Y52F/Y73F double mutant of phospholipase A2: increased hydrophobic interactions of the phenyl groups compensate for the disrupted hydrogen bonds of the tyrosines.

The enzyme phospholipase A2 (PLA2) catalyzes the hydrolysis of the sn-2 ester bond of membrane phospholipids. The highly conserved Tyr residues 52 and 73 in the enzyme form hydrogen bonds to the carboxylate group of the catalytic Asp-99. These hydrogen bonds were initially regarded as essential for the interfacial recognition and the stability of the overall catalytic network. The elimination of the hydrogen bonds involving the phenolic hydroxyl groups of the Tyr-52 and -73 by changing them to Phe lowered the stability but did not significantly affect the catalytic activity of the enzyme. The X-ray crystal structure of the double mutant Y52F/Y73F has been determined at 1.93 A resolution to study the effect of the mutation on the structure. The crystals are trigonal, space group P3(1)21, with cell parameters a = b = 46.3 A and c = 102.95 A. Intensity data were collected on a Siemens area detector, 8,024 reflections were unique with an R(sym) of 4.5% out of a total of 27,203. The structure was refined using all the unique reflections by XPLOR to a final R-factor of 18.6% for 955 protein atoms, 91 water molecules, and 1 calcium ion. The root mean square deviation for the alpha-carbon atoms between the double mutant and wild type was 0.56 A. The crystal structure revealed that four hydrogen bonds were lost in the catalytic network; three involving the tyrosines and one involving Pro-68. However, the hydrogen bonds of the catalytic triad, His-48, Asp-99, and the catalytic water, are retained. There is no additional solvent molecule at the active site to replace the missing hydroxyl groups; instead, the replacement of the phenolic OH groups by H atoms draws the Phe residues closer to the neighboring residues compared to wild type; Phe-52 moves toward His-48 and Asp-99 of the catalytic diad, and Phe-73 moves toward Met-8, both by about 0.5 A. The closing of the voids left by the OH groups increases the hydrophobic interactions compensating for the lost hydrogen bonds. The conservation of the triad hydrogen bonds and the stabilization of the active site by the increased hydrophobic interactions could explain why the double mutant has activity similar to wild type. The results indicate that the aspartyl carboxylate group of the catalytic triad can function alone without additional support from the hydrogen bonds of the two Tyr residues.

Electrostatic complementarity in molecular associations

In this paper, I attempt to summarize the main qualitative features of electrostatic complementarity and similarity, important determinants of molecular recognition. The two aspects, Coulombic and hydrophobic matching, can be formulated in terms of molecular electrostatic potentials and fields. The Coulombic aspect is equivalent to the requirement to produce a potential pattern in the host cavity that is opposite in sign to that emerging from a guest. Hydrophobic complementarity is best described by the similis simili gaudet principle. This means that field patterns near the interacting molecular surfaces must be of similar magnitude. The above rules, which may find useful application in molecular graphics, were studied for different cases of enzyme-ligand interactions in trypsin. A further example, a noncovalent structural model of the catalytic diad in Streptomyces Griseus protease A, supports the observation that the same molecular entities form similar associations even in different environments, as is the case in the complex of small species in a crystal and amino acid residues with structural water molecules in a protein.