Identification of the histidine and aspartic acid residues essential for enzymatic activity of a family I.3 lipase by site-directed mutagenesis

A lipase from Pseudomonas sp. MIS38 (PML) is a member of the lipase family I.3. We analyzed the roles of the five histidine residues (His(30), His(274), His(291), His(313), and His(365)) and five acidic amino acid residues (Glu(253), Asp(255), Asp(262), Asp(275), and Asp(290)), which are fully conserved in the amino acid sequences of family I.3 lipases, by site-directed mutagenesis. We showed that the mutation of His(313) or Asp(255) to Ala almost fully inactivated the enzyme, whereas the mutations of other residues to Ala did not seriously affect the enzymatic activity. Measurement of the far- and near-UV circular dichroism spectra suggests that inactivation by the mutation of His(313) or Asp(255) is not due to marked changes in the tertiary structure. We propose that His(313) and Asp(255), together with Ser(207), form a catalytic triad in PML.     

Homology modeling and identification of serine 160 as nucleophile of the active site in a thermostable carboxylesterase from the archaeon Archaeoglobus fulgidus

The hyperthermophilic Archaeon Archaeoglobus fulgidus has a gene (AF1763) which encodes a thermostable carboxylesterase belonging to the hormone-sensitive lipase (HSL)-like group of the esterase/lipase family. Based on secondary structure predictions and a secondary structure-driven multiple sequence alignment with remote homologous proteins of known three-dimensional structure, we previously hypothesized for this enzyme the alpha/beta-hydrolase fold typical of several lipases and esterases and identified Ser160, Asp 255 and His285 as the putative members of the catalytic triad. In this paper we report the building of a 3D model for this enzyme based on the structure of the homologous brefeldin A esterase from Bacillus subtilis whose structure has been recently elucidated. The model reveals the topological organization of the fold corroborating our predictions. As regarding the active-site residues, Ser160, Asp255 and His285 are located close each other at hydrogen bond distances. The catalytic role of Ser160 as the nucleophilic member of the triad is demonstrated by the [(3)H]diisopropylphosphofluoridate (DFP) active-site labeling and sequencing of a radioactive peptide containing the signature sequence GDSAGG.     

Crystallographic and kinetic investigations on the mechanism of 6-pyruvoyl tetrahydropterin synthase

The enzyme 6-pyruvoyl tetrahydropterin synthase (PTPS) catalyses the second step in the de novo biosynthesis of tetrahydrobiopterin, the conversion of dihydroneopterin triphosphate to 6-pyruvoyl tetrahydropterin. The Zn and Mg-dependent reaction includes a triphosphate elimination, a stereospecific reduction of the N5-C6 double bond and the oxidation of both side-chain hydroxyl groups. The crystal structure of the inactive mutant Cys42Ala of PTPS in complex with its natural substrate dihydroneopterinetriphosphate was determined at 1.9 A resolution. Additionally, the uncomplexed enzyme was refined to 2.0 A resolution. The active site of PTPS consists of the pterin-anchoring Glu A107 neighboured by two catalytic motifs: a Zn(II) binding site and an intersubunit catalytic triad formed by Cys A42, Asp B88 and His B89. In the free enzyme the Zn(II) is in tetravalent co-ordination with three histidine ligands and a water molecule. In the complex the water is replaced by the two substrate side-chain hydroxyl groups yielding a penta-co-ordinated Zn(II) ion. The Zn(II) ion plays a crucial role in catalysis. It activates the protons of the substrate, stabilizes the intermediates and disfavours the breaking of the C1'C2' bond in the pyruvoyl side-chain. Cys A42 is activated by His B89 and Asp B88 for proton abstraction from the two different substrate side-chain atoms C1', and C2'. Replacing Ala A42 in the mutant structure by the wild-type Cys by modelling shows that the C1' and C2' substrate side-chain protons are at equal distances to Cys A42 Sgamma. The basicity of Cys A42 may be increased by a catalytic triad His B89 and Asp B88. The active site of PTPS seems to be optimised to carry out proton abstractions from two different side-chain C1' and C2' atoms, with no obvious preference for one of them. Kinetic studies with dihydroneopterin monophosphate reveal that the triphosphate moiety of the substrate is necessary for enzyme specifity.     

Comparative site-directed mutagenesis in the catalytic amino acid triad in calicivirus proteases

Calicivirus proteases cleave the viral precursor polyprotein encoded by open reading frame 1 (ORF1) into multiple intermediate and mature proteins. These proteases have conserved histidine (His), glutamic acid (Glu) or aspartic acid (Asp), and cysteine (Cys) residues that are thought to act as a catalytic triad (i.e. general base, acid and nucleophile, respectively). However, is the triad critical for processing the polyprotein? In the present study, we examined these amino acids in viruses representing the four major genera of Caliciviridae: Norwalk virus (NoV), Rabbit hemorrhagic disease virus (RHDV), Sapporo virus (SaV) and Feline calicivirus (FCV). Using single amino‐acid substitutions, we found that an acidic amino acid (Glu or Asp), as well as the His and Cys in the putative catalytic triad, cannot be replaced by Ala for normal processing activity of the ORF1 polyprotein in vitro. Similarly, normal activity is not retained if the nucleophile Cys is replaced with Ser. These results showed the calicivirus protease is a Cys protease and the catalytic triad formation is important for protease activity. Our study is the first to directly compare the proteases of the four representative calicivirus genera. Interestingly, we found that RHDV and SaV proteases critically need the acidic residues during catalysis, whereas proteolytic cleavage occurs normally at several cleavage sites in the ORF1 polyprotein without a functional acid residue in the NoV and FCV proteases. Thus, the substrate recognition mechanism may be different between the SaV and RHDV proteases and the NoV and FCV proteases.     

Mutation analysis of carbamoyl phosphate synthetase: Does the structurally conserved glutamine amidotransferase triad act as a functional dyad?

Evolutionarily conserved triad glutamine amidotransferase (GAT) domains catalyze the cleavage of glutamine to yield ammonia and sequester the ammonia in a tunnel until delivery to a variety of acceptor substrates in synthetase domains of variable structure. Whereas a conserved hydrolytic triad (Cys/His/Glu) is observed in the solved GAT structures, the specificity pocket for glutamine is not apparent, presumably because its formation is dependent on the conformational change that couples acceptor availability to a greatly increased rate of glutamine cleavage. In Escherichia coli carbamoyl phosphate synthetase (eCPS), one of the best characterized triad GAT members, the Cys269 and His353 triad residues are essential for glutamine hydrolysis, whereas Glu355 is not critical for eCPS activity. To further define the glutamine-binding pocket and possibly identify an alternative member of the catalytic triad that is situated for this role in the coupled conformation, we have analyzed mutations at Gln310, Asn311, Asp334, and Gln351, four conserved, but not yet analyzed residues that might potentially function as the third triad member. Alanine substitution of Gln351, Asn311, and Gln310 yielded respective K(m) increases of 145, 27, and 15, suggesting that Gln351 plays a key role in glutamine binding in the coupled conformation, and that Asn311 and Gln310 make less significant contributions. None of the mutant k (cat) values varied significantly from those for wild-type eCPS. Combined with previously reported data on other conserved eCPS residues, these results strongly suggest that Cys269 and His353 function as a catalytic dyad in the GAT site of eCPS.     

Structure determinants and substrate recognition of serine carboxypeptidase-like acyltransferases from plant secondary metabolism

Structures of the serine carboxypeptidase-like enzymes 1-O-sinapoyl-beta-glucose:L-malate sinapoyltransferase (SMT) and 1-O-sinapoyl-beta-glucose:choline sinapoyltransferase (SCT) were modeled to gain insight into determinants of specificity and substrate recognition. The structures reveal the alpha/beta-hydrolase fold as scaffold for the catalytic triad Ser-His-Asp. The recombinant mutants of SMT Ser173Ala and His411Ala were inactive, whereas Asp358Ala displayed residual activity of 20%. 1-O-sinapoyl-beta-glucose recognition is mediated by a network of hydrogen bonds. The glucose moiety is recognized by a hydrogen bond network including Trp71, Asn73, Glu87 and Asp172. The conserved Asp172 at the sequence position preceding the catalytic serine meets sterical requirements for the glucose moiety. The mutant Asn73Ala with a residual activity of 13% underscores the importance of the intact hydrogen bond network. Arg322 is of key importance by hydrogen bonding of 1-O-sinapoyl-beta-glucose and L-malate. By conformational change, Arg322 transfers L-malate to a position favoring its activation by His411. Accordingly, the mutant Arg322Glu showed 1% residual activity. Glu215 and Arg219 establish hydrogen bonds with the sinapoyl moiety. The backbone amide hydrogens of Gly75 and Tyr174 were shown to form the oxyanion hole, stabilizing the transition state. SCT reveals also the catalytic triad and a hydrogen bond network for 1-O-sinapoyl-beta-glucose recognition, but Glu274, Glu447, Thr445 and Cys281 are crucial for positioning of choline.     

Cysteine proteases of positive strand RNA viruses and chymotrypsin-like serine proteases. A distinct protein superfamily with a common structural fold

Evidence is presented, based on sequence comparison and secondary structure prediction, of structural and evolutionary relationship between chymotrypsin-like serine proteases, cysteine proteases of positive strand RNA viruses (3C proteases of picornaviruses and related enzymes of como-, nepo- and potyviruses) and putative serine protease of a sobemovirus. These observations lead to re-identification of principal catalytic residues of viral proteases. Instead of the pair of Cys and His, both located in the C-terminal part of 3C proteases, a triad of conserved His, Asp(Glu) and Cys(Ser) has been identified, the first two residues resident in the N-terminal, and Cys in the C-terminal beta-barrel domain. These residues are suggested to form a charge-transfer system similar to that formed by the catalytic triad of chymotrypsin-like proteases. Based on the structural analogy with chymotrypsin-like proteases, the His residue previously implicated in catalysis, together with two partially conserved Gly residues, is predicted to constitute part of the substrate-binding pocket of 3C proteases. A partially conserved ThrLys/Arg dipeptide located in the loop preceding the catalytic Cys is suggested to confer the primary cleavage specificity of 3C toward Glx/Gly(Ser) sites. These observations provide the first example of relatedness between proteases belonging, by definition, to different classes.     

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.     

A novel engineered subtilisin BPN' lacking a low-barrier hydrogen bond in the catalytic triad

The low-barrier hydrogen bond (LBHB) between the Asp and His residues of the catalytic triad in a serine protease was perturbed via the D32C mutation in subtilisin BPN' (Bacillus protease N'). This mutant enzyme catalyzes the hydrolysis of N-Suc-Ala-Ala-Pro-Phe-SBzl with a k(cat)/K(m) value that is only 8-fold reduced from that of the wild-type (WT) enzyme. The value of k(cat)/K(m) for the corresponding p-nitroanilide (pNA) substrate is only 50-fold lower than that of the WT enzyme (DeltaDeltaG++ = 2.2 kcal/mol). The pK(a) controlling the ascending limb of the pH versus k(cat)/K(m) profile is lowered from 7.01 (WT) to 6.53 (D32C), implying that any hydrogen bond replacing that between Asp32 and His64 of the WT enzyme most likely involves the neutral thiol rather than the thiolate form of Cys32. It is shown by viscosity variation that the reaction of WT subtilisin with N-Suc-Ala-Ala-Pro-Phe-SBzl is 50% (sucrose) to 100% (glycerol) diffusion-controlled, while that of the D32C construct is 29% (sucrose) to 76% (glycerol) diffusion-controlled. The low-field NMR resonance of 18 ppm that has been assigned to a proton shared by Asp32 and His64, and is considered diagnostic of a LBHB in the WT enzyme, is not present in D32C subtilisin. Thus, the LBHB is not an inherent requirement for substantial rate enhancement for subtilisin.     

Identification of the catalytic residues of alpha-amino acid ester hydrolase from Acetobacter turbidans by labeling and site-directed mutagenesis

The alpha-amino acid ester hydrolase from Acetobacter turbidans ATCC 9325 is capable of hydrolyzing and synthesizing the side chain peptide bond in beta-lactam antibiotics. Data base searches revealed that the enzyme contains an active site serine consensus sequence Gly-X-Ser-Tyr-X-Gly that is also found in X-prolyl dipeptidyl aminopeptidase. The serine hydrolase inhibitor p-nitrophenyl-p'-guanidino-benzoate appeared to be an active site titrant and was used to label the alpha-amino acid ester hydrolase. Electrospray mass spectrometry and tandem mass spectrometry analysis of peptides from a CNBr digest of the labeled protein showed that Ser(205), situated in the consensus sequence, becomes covalently modified by reaction with the inhibitor. Extended sequence analysis showed alignment of this Ser(205) with the catalytic nucleophile of some alpha/beta-hydrolase fold enzymes, which posses a catalytic triad composed of a nucleophile, an acid, and a base. Based on the alignments, 10 amino acids were selected for site-directed mutagenesis (Arg(85), Asp(86), Tyr(143), Ser(156), Ser(205), Tyr(206), Asp(338), His(370), Asp(509), and His(610)). Mutation of Ser(205), Asp(338,) or His(370) to an alanine almost fully inactivated the enzyme, whereas mutation of the other residues did not seriously affect the enzyme activity. Circular dichroism measurements showed that the inactivation was not caused by drastic changes in the tertiary structure. Therefore, we conclude that the catalytic domain of the alpha-amino acid ester hydrolase has an alpha/beta-hydrolase fold structure with a catalytic triad of Ser(205), Asp(338), and His(370). This distinguishes the alpha-amino acid ester hydrolase from the Ntn-hydrolase family of beta-lactam antibiotic acylases.     

Three-dimensional model of a substrate-bound SARS chymotrypsin-like cysteine proteinase predicted by multiple molecular dynamics simulations: catalytic efficiency regulated by substrate binding

Severe acute respiratory syndrome (SARS) is a contagious and deadly disease caused by a new coronavirus. The protein sequence of the chymotrypsin-like cysteine proteinase (CCP) responsible for SARS viral replication has been identified as a target for developing anti-SARS drugs. Here, I report the ATVRLQ(p1)A(p1')-bound CCP 3D model predicted by 420 different molecular dynamics simulations (2.0 ns for each simulation with a 1.0-fs time step). This theoretical model was released at the Protein Data Bank (PDB; code: 1P76) before the release of the first X-ray structure of CCP (PDB code: 1Q2W). In contrast to the catalytic dyad observed in X-ray structures of CCP and other coronavirus cysteine proteinases, a catalytic triad comprising Asp187, His41, and Cys145 is found in the theoretical model of the substrate-bound CCP. The simulations of the CCP complex suggest that substrate binding leads to the displacement of a water molecule entrapped by Asp187 and His41, thus converting the dyad to a more efficient catalytic triad. The CCP complex structure has an expanded active-site pocket that is useful for anti-SARS drug design. In addition, this work demonstrates that multiple molecular dynamics simulations are effective in correcting errors that result from low-sequence-identity homology modeling.     

Breaking the low barrier hydrogen bond in a serine protease

The serine protease subtilisin BPN' is a useful catalyst for peptide synthesis when dissolved in high concentrations of a water-miscible organic co-solvent such as N,N-dimethylformamide (DMF). However, in 50% DMF, the k(cat) for amide hydrolysis is two orders of magnitude lower than in aqueous solution. Surprisingly, the k(cat) for ester hydrolysis is unchanged in 50% DMF. To explain this alteration in activity, the structure of subtilisin 8397+1 was determined in 20, 35, and 50% (v/v) DMF to 1.8 A resolution. In 50% DMF, the imidazole ring of His64, the central residue of the catalytic triad, has rotated approximately 180 degrees around the Cbeta-Cgamma bond. Two new water molecules in the active site stabilize the rotated conformation. This rotation places His64 in an unfavorable geometry to interact with the other members of the catalytic triad, Ser221 and Asp32. NMR experiments confirm that the characteristic resonance due to the low barrier hydrogen bond between the His64 and Asp32 is absent in 50% DMF. These experiments provide a clear structural basis for the change in activity of serine proteases in organic co-solvents.     

Structural relationship between lipases and peptidases of the prolyl oligopeptidase family.

In prolyl oligopeptidase and its homologues, which constitute a new serine protease family, the order of the catalytic Ser and His residues in the amino acid sequence is the reverse of what is found in the trypsin and subtilisin families. The exact position of the third member of the catalytic triad, an Asp residue, has not yet been identified in the new family. Recent determination of the three-dimensional structures of pancreatic and microbial lipases has shown that the order of their catalytic residues is Ser, Asp, His, and this fits the order Ser, His of prolyl oligopeptidase. However, there is no sequence homology between lipases and peptidases, except for a 10-residue segment, which encompasses the essential Ser, and for the immediate vicinity of the catalytic Asp and His residues. This comparison identifies the catalytic Asp residue in the prolyl oligopeptidase family. The relative positions of the three catalytic residues in peptidases and microbial lipases were the same and this indicated structural and possibly evolutionary relationship between the two families.     

Structural and mutational characterization of the catalytic A-module of the mannuronan C-5-epimerase AlgE4 from Azotobacter vinelandii

Alginate is a family of linear copolymers of (1-->4)-linked beta-d-mannuronic acid and its C-5 epimer alpha-l-guluronic acid. The polymer is first produced as polymannuronic acid and the guluronic acid residues are then introduced at the polymer level by mannuronan C-5-epimerases. The structure of the catalytic A-module of the Azotobacter vinelandii mannuronan C-5-epimerase AlgE4 has been determined by x-ray crystallography at 2.1-A resolution. AlgE4A folds into a right-handed parallel beta-helix structure originally found in pectate lyase C and subsequently in several polysaccharide lyases and hydrolases. The beta-helix is composed of four parallel beta-sheets, comprising 12 complete turns, and has an amphipathic alpha-helix near the N terminus. The catalytic site is positioned in a positively charged cleft formed by loops extending from the surface encompassing Asp(152), an amino acid previously shown to be important for the reaction. Site-directed mutagenesis further implicates Tyr(149), His(154), and Asp(178) as being essential for activity. Tyr(149) probably acts as the proton acceptor, whereas His(154) is the proton donor in the epimerization reaction.     

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.     

Crystal structure of pseudomonas aeruginosa lipase in the open conformation. The prototype for family I.1 of bacterial lipases

The x-ray structure of the lipase from Pseudomonas aeruginosa PAO1 has been determined at 2.54 A resolution. It is the first structure of a member of homology family I.1 of bacterial lipases. The structure shows a variant of the alpha/beta hydrolase fold, with Ser(82), Asp(229), and His(251) as the catalytic triad residues. Compared with the "canonical" alpha/beta hydrolase fold, the first two beta-strands and one alpha-helix (alphaE) are not present. The absence of helix alphaE allows the formation of a stabilizing intramolecular disulfide bridge. The loop containing His(251) is stabilized by an octahedrally coordinated calcium ion. On top of the active site a lid subdomain is in an open conformation, making the catalytic cleft accessible from the solvent region. A triacylglycerol analogue is covalently bound to Ser(82) in the active site, demonstrating the position of the oxyanion hole and of the three pockets that accommodate the sn-1, sn-2, and sn-3 fatty acid chains. The inhibited enzyme can be thought to mimic the structure of the tetrahedral intermediate that occurs during the acylation step of the reaction. Analysis of the binding mode of the inhibitor suggests that the size of the acyl pocket and the size and interactions of the sn-2 binding pocket are the predominant determinants of the regio- and enantio-preference of the enzyme.     

Crystal structure of a deubiquitinating enzyme (human UCH-L3) at 1.8 A resolution.

Ubiquitin C-terminal hydrolases catalyze the removal of adducts from the C-terminus of ubiquitin. We have determined the crystal structure of the recombinant human Ubiquitin C-terminal Hydrolase (UCH-L3) by X-ray crystallography at 1.8 A resolution. The structure is comprised of a central antiparallel beta-sheet flanked on both sides by alpha-helices. The beta-sheet and one of the helices resemble the well-known papain-like cysteine proteases, with the greatest similarity to cathepsin B. This similarity includes the UCH-L3 active site catalytic triad of Cys95, His169 and Asp184, and the oxyanion hole residue Gln89. Papain and UCH-L3 differ, however, in strand and helix connectivity, which in the UCH-L3 structure includes a disordered 20 residue loop (residues 147-166) that is positioned over the active site and may function in the definition of substrate specificity. Based upon analogy with inhibitor complexes of the papain-like enzymes, we propose a model describing the binding of ubiquitin to UCH-L3. The UCH-L3 active site cleft appears to be masked in the unliganded structure by two different segments of the enzyme (residues 9-12 and 90-94), thus implying a conformational change upon substrate binding and suggesting a mechanism to limit non-specific hydrolysis.     

Proposed mechanism for the condensation reaction of citrate synthase: 1.9-A structure of the ternary complex with oxaloacetate and carboxymethyl coenzyme A

The crystal structure of the ternary complex citrate synthase-oxaloacetate-carboxymethyl coenzyme A has been solved to a resolution of 1.9 A and refined to a conventional crystallographic R factor of 0.185. The structure resembles a proposed transition state of the condensation reaction and suggests that the condensation reaction proceeds through a neutral enol rather than an enolate intermediate. A mechanism for the condensation reaction is proposed which involves the participation of three key catalytic groups (Asp 375, His 274, and His 320) in two distinct steps. The proposed mechanism invokes concerted general acid-base catalysis twice to explain both the energetics of the reaction and the experimentally observed inversion of stereochemistry at the attacking carbon atom.     

Catalytic antibody light chain capable of cleaving a chemokine receptor CCR-5 peptide with a high reaction rate constant

A monoclonal antibody (MAb), ECL2B-2, was obtained by immunizing a peptide possessing a part of a sequence of a chemokine receptor, CCR-5, which is present as a membrane protein on the macrophage surface, and which plays an important role in human immunodeficiency virus (HIV) infection. From the DNA and the deduced amino acid sequences of the light and heavy chains of ECL2B-2 MAb, molecular modeling was conducted to calculate the steric conformation of the antibody. Modeling suggested that the structure of ECL2B-2 could possess one or two catalytic triad(s), composed of Asp(1), Ser(27a) (or Ser(27e)), and His(93) (or His(27d)), in the light chain of ECL2B-2. The three amino acid residues, Asp(1), Ser(27a), and His(93), are identical to those of catalytic antibody light chains such as VIPase and i41SL1-2. The light chain of ECL2B-2 MAb degraded the antigenic peptide CCR-5 within about 100 h. Surprisingly, the light chain had a very high catalytic reaction rate constant (k(cat)) of 2.23 min(-1), which is greater by factors of tens to hundreds than those of natural catalytic antibodies obtained previously. The heavy chain of ECL2B-2 MAb, which has no catalytic triad because of a lack of His residue, did not degrade the CCR-5 peptide.     

A cysteine-histidine-aspartate catalytic triad is involved in glutamine amide transfer function in purF-type glutamine amidotransferases

A family of four glutamine amidotransferases has a homologous glutamine amide transfer domain, designated purF-type, that is named after purF-encoded glutamine phosphoribosylpyrophosphate amidotransferase. The glutamine amide transfer domain of approximately 194 amino acid residues is at the NH2 terminus of the protein chain. Site-directed mutagenesis was used to replace several of the 9 invariant amino acids in the glutamine amide transfer domain of glutamine phosphoribosylpyrophosphate amidotransferase. The results indicate that a Cys1-His101-Asp29 catalytic triad is involved in the glutamine amide transfer function of this enzyme. The evidence suggests that His101 functions to increase the nucleophilicity of Cys1, which is used to form a glutamine-enzyme covalent intermediate. Asp29 has a role subsequent to formation of the covalent intermediate. The Cys-His-Asp catalytic triad is implicated in the glutamine amide transfer function of purF-type amidotransferases.