Alpha/beta‐hydrolases: A unique structural motif coordinates catalytic acid residue in 40 protein fold families

The alpha/beta‐hydrolases are a family of acid‐base‐nucleophile catalytic triad enzymes with a common fold, but using a wide variety of substrates, having different pH optima, catalyzing unique catalytic reactions and often showing improved chemical and thermo stability. The ABH enzymes are prime targets for protein engineering. Here, we have classified active sites from 51 representative members of 40 structural ABH fold families into eight distinct conserved geometries. We demonstrate the occurrence of a common structural motif, the catalytic acid zone, at the catalytic triad acid turn. We show that binding of an external ligand does not change the structure of the catalytic acid zone and both the ligand‐free and ligand‐bound forms of the protein belong to the same catalytic acid zone subgroup. We also show that the catalytic acid zone coordinates the position of the catalytic histidine loop directly above its plane, and consequently, fixes the catalytic histidine in a proper position near the catalytic acid. Finally, we demonstrate that the catalytic acid zone plays a key role in multi‐subunit complex formation in ABH enzymes, and is involved in interactions with other proteins. As a result, we speculate that each of the catalytic triad residues has its own supporting structural scaffold, similar to the catalytic acid zone, described above, which together form the extended catalytic triad motif. Each scaffold coordinates the function of its respective catalytic residue, and can even compensate for the loss of protein function, if the catalytic amino acid is mutated.     

催化性抗体及其研究进展

根据过渡态理论,按特定的化学反应机制确定反应中的可能过渡态结构,选择和该过渡态结构类似的化合物作为半抗原,可诱导机体产生具有催化活性的催化性抗体.文章对诱导催化性抗体中半抗原的选择原则、催化性抗体和非催化性抗体间的联系、催化性抗体和酶促催化反应的比较等方面进行了较为全面的综述,并对催化性抗体在医药科学中的应用前景及限制因素进行了讨论.     

Chiral Symmetry Breaking in Large Peptide Systems

Chiral symmetry breaking in far from equilibrium systems with large number of amino acids and peptides, like a prebiotic Earth, was considered. It was shown that if organic catalysts were abundant, then effective averaging of enantioselectivity would prohibit any symmetry breaking in such systems. It was further argued that non-linear (catalytic) reactions must be very scarce (called the abundance parameter) and catalysts should work on small groups of similar reactions (called the similarity parameter) in order to chiral symmetry breaking have a chance to occur. Models with 20 amino acids and peptide lengths up to three were considered. It was shown that there are preferred ranges of abundance and similarity parameters where the symmetry breaking can occur in the models with catalytic synthesis / catalytic destruction / both catalytic synthesis and catalytic destruction. It was further shown that models with catalytic synthesis and catalytic destruction statistically result in a substantially higher percentage of the models where the symmetry breaking can occur in comparison to the models with just catalytic synthesis or catalytic destruction. It was also shown that when chiral symmetry breaking occurs, then concentrations of some amino acids, which collectively have some mutually beneficial properties, go up, whereas the concentrations of the ones, which don’t have such properties, go down. An open source code of the whole system was provided to ensure that the results can be checked, repeated, and extended further if needed.

     

Constant Domain-regulated Antibody Catalysis

Some antibodies contain variable (V) domain catalytic sites. We report the superior amide and peptide bond-hydrolyzing activity of the same heavy and light chain V domains expressed in the IgM constant domain scaffold compared with the IgG scaffold. The superior catalytic activity of recombinant IgM was evident using two substrates, a small model peptide that is hydrolyzed without involvement of high affinity epitope binding, and HIV gp120, which is recognized specifically by noncovalent means prior to the hydrolytic reaction. The catalytic activity was inhibited by an electrophilic phosphonate diester, consistent with a nucleophilic catalytic mechanism. All 13 monoclonal IgMs tested displayed robust hydrolytic activities varying over a 91-fold range, consistent with expression of the catalytic functions at distinct levels by different V domains. The catalytic activity of polyclonal IgM was superior to polyclonal IgG from the same sera, indicating that on average IgMs express the catalytic function at levels greater than IgGs. The findings indicate a favorable effect of the remote IgM constant domain scaffold on the integrity of the V-domain catalytic site and provide a structural basis for conceiving antibody catalysis as a first line immune function expressed at high levels prior to development of mature IgG class antibodies.     

Pathogenicity of catalytic antibodies: catalytic activity of Bence Jones proteins from myeloma patients with renal impairment can elicit cytotoxic effects

Some Bence Jones proteins (BJPs) can display catalytic activity. Although the catalytic activity of BJPs might be associated with the pathogenesis of disease, this relationship has not yet been established. We tested the effects of seven BJPs on LLC-PK1 cells to assess their pathogenicity. Two out of the seven BJPs showed cytotoxic activity, as assessed by microscopic analysis, the WST method and TUNEL staining. Moreover, the cytotoxic BJPs were excreted by patients who presented with renal impairment. The cytotoxic BJPs displayed 20- to 40-fold higher catalytic activities (kcat of 3.5-2.2 min(-1)) in hydrolyzing a chromogenic substrate compared to the other BJPs. By treating the cytotoxic BJPs with diisopropylfluorophosphate, they lost not only their catalytic activity, but also the cytotoxic effects. These results indicate a direct link between cytotoxicity and the catalytic activity of the BJPs. The catalytic activity of BJPs contributes to the pathogenesis, as well as to development, of symptoms of multiple myeloma. Inhibition of the catalytic activity of BJPs may form the basis of a novel treatment for multiple myeloma patients with renal dysfunction.     

Different cleavage specificities of the dual catalytic domains in chitinase from the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1

The chitinase from the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1, Tk-ChiA, has an interesting multidomain structure containing dual catalytic domains and triple chitin-binding domains. To determine the biochemical properties of each domain, we constructed deletion mutant genes corresponding to the individual catalytic domains and purified the recombinant proteins. A synergistic effect was observed when chitin was degraded in the presence of both catalytic domains, suggesting different cleavage specificity of these domains. Analyses of degradation products from N-acetyl-chitooligosaccharides and their chromogenic derivatives with thin layer chromatography indicated that the N-terminal catalytic domain mainly hydrolyzed the second glycosidic bond from the nonreducing end of the oligomers, whereas the C-terminal domain randomly hydrolyzed glycosidic bonds other than the first bond from the nonreducing end. Both catalytic domains formed diacetyl-chitobiose as a major end product and possessed transglycosylation activity. Further analysis of degradation products from colloidal chitin with high performance liquid chromatography showed that the N-terminal catalytic domain exclusively liberated diacetyl-chitobiose, whereas reactions with the C-terminal domain led to N-acetyl-chitooligosaccharides of various lengths. These results demonstrated that the N-terminal and C-terminal catalytic domains functioned as exo- and endochitinases, respectively. The biochemical results provide a physiological explanation for the presence of two catalytic domains with different specificity and suggest a cooperative function between the two on a single polypeptide in the degradation of chitin.     

A novel combination of two classic catalytic schemes

The crystal structure of an alkaline Bacillus cellulase catalytic core, from glucoside hydrolase family 5, reveals a novel combination of the catalytic machinery of two classic textbook enzymes. The enzyme has the expected two glutamate residues in close proximity to one another in the active-site that are typical of retaining cellulases. However, the proton donor, glutamate 139 is also unexpectedly a member of a serine-histidine-glutamate catalytic triad, forming a novel combination of catalytic machineries. Structure and sequence analysis of glucoside hydrolase family 5 reveal that the triad is highly conserved, but with variations at the equivalent of the serine position. We speculate that the purpose of this novel catalytic triad is to control the protonation of the acid/base glutamate, facilitating the first step of the catalytic reaction, protonation of the substrate, by the proton donor glutamate. If correct, this will be a novel use for a catalytic triad.     

The defective proton-ATPase of uncD mutants of Escherichia coli. Two mutations which affect the catalytic mechanism

The catalytic characteristics of F1-ATPases from uncD412 and uncD484 mutant strains of Escherichia coli were studied in order to understand how these beta-subunit mutations cause defective catalysis. Both mutant enzymes showed reduced affinity for ATP at the first catalytic site. While uncD412 F1 was similar to normal in other aspects of single site catalysis, uncD484 F1 showed a Keq of bound reactants greatly biased toward bound substrate ATP and an abnormally fast rate of Pi release. Impairment of productive catalytic cooperativity was the major cause of the reduced steady state ("multisite") catalytic rate in both mutant enzymes. Addition of excess ATP to saturate second and/or third catalytic sites did promote ATP hydrolysis and product release at the first catalytic site of uncD412 F1, but the multisite turnover rate was significantly slower than normal. In contrast, with uncD484 F1, addition of excess ATP induced rapid release of ATP from the first catalytic site and so productive catalytic cooperativity was almost completely absent. The results show that both mutations affect properties of the catalytic site and catalytic site cooperativity and further that the relatively more severe uncD484 mutation affects a residue which acts as a determinant of the fate of bound substrate ATP during promotion of catalysis. Taken together with previous studies of uncA mutant F1-ATPases (Wise, J. G., Latchney, L. R., Ferguson, A. M., and Senior, A. E. (1984) Biochemistry 23, 1426-1432) the results indicate that catalytic site cooperativity in F1-ATPases involves concerted beta-alpha-beta intersubunit communication between catalytic sites on the beta-subunits.     

Dispensable pieces of an aminoacyl tRNA synthetase which activate the catalytic site

Recent data suggest that size polymorphism of aminoacyl tRNA synthetase is due to variable fusions of additional functional domains to a catalytic core so that, in a large synthetase, a substantial part of the polypeptide is dispensable for catalytic activity. We demonstrate here that a dispensable domain, joined to the catalytic core of a large synthetase, can activate the catalytic sites. This is shown by complementation of an activity-deficient mutant enzyme by protein fragments that contain internal deletions within the catalytic domain and are themselves devoid of activity. The complementation is dependent upon the presence of a defined segment of polypeptide that is remote in the sequence from the catalytic core. Substantial coupling has been established between dispensable and indispensable component pieces. This could be a mechanism to build efficiently large enzymes which integrate the catalytic sites with other previously shown functional roles.     

Intermediates in the refolding of ribonuclease at subzero temperatures. 2. Monitoring by inhibitor binding and catalytic activity

The kinetics of refolding of ribonuclease A were monitored by the return of catalytic activity and inhibitor binding at -15 degrees C in 35% methanol cryosolvent at pH* 3.0 and 6.0. Catalytic activity was measured with cytidine 2',3'-cyclic monophosphate as substrate; inhibitor binding was determined with the competitive inhibitor cytidine 2'-monophosphate. Biphasic kinetics were observed at pH* 3.0 for both return of catalytic activity and inhibitor binding. At pH* 6.0 the rate of return of catalytic activity was monophasic, whereas that of inhibitor binding was biphasic. For both inhibitor binding and catalytic activity one of the observed rates was pH-dependent. Full return of catalytic activity was obtained at the completion of the refolding process. The observations are interpreted in terms of two parallel pathways of refolding for slow-refolding ribonuclease, with several native-like, partially folded intermediate states on the minor slow-refolding pathway. Of particular note is the presence of at least one such species that has inhibitor-binding capacity but not catalytic activity. This may be rationalized in terms of the known native structure. In addition, an intermediate is postulated which has the incorrect Pro-93 conformation and only partial catalytic activity (42% of the native). The slowest observed transient is attributed to the isomerization of this proline residue and return of full catalytic activity.     

Structural basis for the dynamics of human methionyl-tRNA synthetase in multi-tRNA synthetase complexes

In mammals, eight aminoacyl-tRNA synthetases (AARSs) and three AARS-interacting multifunctional proteins (AIMPs) form a multi-tRNA synthetase complex (MSC). MSC components possess extension peptides for MSC assembly and specific functions. Human cytosolic methionyl-tRNA synthetase (MRS) has appended peptides at both termini of the catalytic main body. The N-terminal extension includes a glutathione transferase (GST) domain responsible for interacting with AIMP3, and a long linker peptide between the GST and catalytic domains. Herein, we determined crystal structures of the human MRS catalytic main body, and the complex of the GST domain and AIMP3. The structures reveal human-specific structural details of the MRS, and provide a dynamic model for MRS at the level of domain orientation. A movement of zinc knuckles inserted in the catalytic domain is required for MRS catalytic activity. Depending on the position of the GST domain relative to the catalytic main body, MRS can either block or present its tRNA binding site. Since MRS is part of a huge MSC, we propose a dynamic switching between two possible MRS conformations; a closed conformation in which the catalytic domain is compactly attached to the MSC, and an open conformation with a free catalytic domain dissociated from other MSC components.     

Regulation of CO production in cerebral microvessels of newborn pigs

Carbon monoxide (CO) is produced from heme by heme oxygenase-2 (HO-2) in cerebral blood vessels. Gas chromatography-mass spectrometry was used on piglet cerebral microvessels to address the hypothesis that CO production is regulated by heme delivery and HO-2 catalytic activity. CO production appears to be substrate limited because heme and its precursor aminolevulinate increase CO production. Ionomycin also increases CO production. However, CO production from exogenous heme was the same in Ca-replete medium, Ca-free medium with ionomycin, and Ca-replete medium with ionomycin. Phorbol myristate acetate increases CO production but does not change the catalytic activity of HO-2. Also, the protein kinase C inhibitor 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine has no effect on the HO-2 catalytic activity. Protein tyrosine kinase inhibition reduces HO-2 catalytic activity. Inhibition of protein tyrosine phosphatases increased HO-2 catalytic activity. Therefore, regulation of CO production by cerebral microvessels can include changing heme availability and HO-2 catalytic activity. HO-2 catalytic activity is stimulated by tyrosine phosphorylation.     

Expression of an active, monomeric catalytic domain of the cGMP-binding cGMP-specific phosphodiesterase (PDE5)

Phosphodiesterases (PDEs) comprise a superfamily of phosphohydrolases that degrade 3',5'-cyclic nucleotides. All known mammalian PDEs are dimeric, but the functional significance of dimerization is unknown. A deletion mutant of cGMP-binding cGMP-specific PDE (PDE5), encoding the 357 carboxyl-terminal amino acids including the catalytic domain, has been generated, expressed, and purified. The K(m) of the catalytic fragment for cGMP (5.5 +/- 0. 51 microM) compares well with those of the native bovine lung PDE5 (5.6 microM) and full-length wild type recombinant PDE5 (2 +/- 0.4 microM). The catalytic fragment and full-length PDE5 have similar IC(50) values for the inhibitors 3-isobutyl-1-methylxanthine (20 microM) and sildenafil (Viagra(TM))(4 nM). Based on measured values for Stokes radius (29 A) and sedimentation coefficient (2.9 S), the PDE5 catalytic fragment has a calculated molecular mass of 35 kDa, which agrees well with that predicted by amino acid content (43.3 kDa) and with that estimated using SDS-polyacrylamide gel electrophoresis (39 kDa). The combined data indicate that the recombinant PDE5 catalytic fragment is monomeric, and retains the essential catalytic features of the dimeric, full-length enzyme. Therefore, the catalytic activity of PDE5 holoenzyme requires neither interaction between the catalytic and regulatory domains nor interactions between subunits of the dimer.     

Chlorophyllase as a serine hydrolase: identification of a putative catalytic triad

Chlorophyllases (Chlases), cloned so far, contain a lipase motif with the active serine residue of the catalytic triad of triglyceride lipases. Inhibitors specific for the catalytic serine residue in serine hydrolases, which include lipases effectively inhibited the activity of the recombinant Chenopodium album Chlase (CaCLH). From this evidence we assumed that the catalytic mechanism of hydrolysis by Chlase might be similar to those of serine hydrolases that have a catalytic triad composed of serine, histidine and aspartic acid in their active site. Thus, we introduced mutations into the putative catalytic residue (Ser162) and conserved amino acid residues (histidine, aspartic acid and cysteine) to generate recombinant CaCLH mutants. The three amino acid residues (Ser162, Asp191 and His262) essential for Chlase activity were identified. These results indicate that Chlase is a serine hydrolase and, by analogy with a plausible catalytic mechanism of serine hydrolases, we proposed a mechanism for hydrolysis catalyzed by Chlase.     

Cloning, sequencing, and expression of a Eubacterium cellulosolvens 5 gene encoding an endoglucanase (Cel5A) with novel carbohydrate-binding modules, and properties of Cel5A

A novel Eubacterium cellulosolvens 5 gene encoding an endoglucanase (Cel5A) was cloned and expressed in Escherichia coli, and its enzymatic properties were characterized. The cel5A gene consists of a 3,444-bp open reading frame and encodes a 1,148-amino-acid protein with a molecular mass of 127,047 Da. Cel5A is a modular enzyme consisting of an N-terminal signal peptide, two glycosyl hydrolase family 5 catalytic modules, two novel carbohydrate-binding modules (CBMs), two linker sequences, and a C-terminal sequence with an unknown function. The amino acid sequences of the two catalytic modules and the two CBMs are 94% and 73% identical to each other, respectively. Two regions that consisted of one CBM and one catalytic module were tandemly connected via a linker sequence. The CBMs did not exhibit significant sequence similarity with any other CBMs. Analyses of the hydrolytic activity of the recombinant Cel5A (rCel5A) comprising the CBMs and the catalytic modules showed that the enzyme is an endoglucanase with activities with carboxymethyl cellulose, lichenan, acid-swollen cellulose, and oat spelt xylan. To investigate the functions of the CBMs and the catalytic modules, truncated derivatives of rCel5A were constructed and characterized. There were no differences in the hydrolytic activities with various polysaccharides or in the hydrolytic products obtained from cellooligosaccharides between the two catalytic modules. Both CBMs had the same substrate affinity with intact rCel5A. Removal of the CBMs from rCel5A reduced the catalytic activities with various polysaccharides remarkably. These observations show that CBMs play an important role in the catalytic function of the enzyme.     

The phosphotransferase activity of casein kinase II is required for its physiological function in vivo.

We have previously shown that the inviability associated with disruption of both catalytic subunits of casein kinase II in Saccharomyces cerevisiae can be rescued by plasmids expressing the catalytic subunit of the Drosophila enzyme (Padmanabha et al., 1990, Mol. Cell. Biol. 10, 4089). Here we describe the construction of mutant forms of the Drosophila catalytic subunit in which residues known to be crucial for catalytic activity in other protein kinases have been altered by site-directed mutagenesis. Mutation of either Lys66 or Asp173, which correspond to Lys72 and Asp184 of cAMP-dependent protein kinase, respectively, yields a casein kinase II catalytic subunit which fails to rescue a yeast strain lacking both endogenous catalytic subunit genes. The data indicate that the phosphotransferase activity of casein kinase II is required for its physiological function in vivo.     

Importance of domain closure for the catalysis and regulation of Escherichia coli aspartate transcarbamoylase

Two hybrid versions of Escherichia coli aspartate transcarbamoylase were studied to determine the influence of domain closure on the homotropic and heterotropic properties of the enzyme. Each hybrid holoenzyme had one wild-type and one inactive catalytic subunit. In the first case the inactive catalytic subunit had Arg-54 replaced by alanine. The holoenzyme with this mutation in all six catalytic chains exhibits a 17,000-fold reduction in activity, no loss in substrate affinity, and an R state structurally identical to that of the wild-type enzyme. In the second case, the inactive catalytic subunit had Arg-105 replaced by alanine. The holoenzyme with this mutation in all six catalytic chains exhibits a 1,100-fold reduction in activity, substantial loss in substrate affinity, and loss of the ability to be converted to the R state. Thus, the R54A substitution results in a holoenzyme that can undergo closure of the catalytic chain domains to form the high activity, high affinity active site and to undergo the allosteric transition, whereas the R105A substitution results in a holoenzyme that can neither undergo domain closure nor the allosteric transition. The hybrid holoenzyme with one wild-type and one R54A catalytic subunit exhibited the same maximal velocity per active site as the wild-type holoenzyme, reduced cooperativity, and normal heterotropic interactions. The hybrid with one wild-type and one R105A catalytic subunit exhibited significantly reduced maximal velocity per active site as compared with the wild-type holoenzyme, reduced cooperativity, and substantially reduced heterotropic interactions. Small angle x-ray scattered was used to verify that the R105A-containing hybrid could attain an R state structure. These results indicate the global nature of the conformational changes associated with the allosteric transition in the enzyme. If one catalytic subunit cannot undergo domain closure to create the active sites, then the entire molecule cannot attain the high activity, high activity R state.     

Cadmium-109 as a probe of the metal binding sites in horse liver alcohol dehydrogenase.

The noncatalytic and catalytic zinc atoms of horse liver alcohol dehydrogenase, [(LADH)Zn2Zn2] or LADH, have been replaced differentially with 109Cd by equilibrium dialysis, resulting in two new enzymatically active species, [(LADH)109Cd2Zn2] and [(LADH)109Cd2109Cd2]. The UV difference spectra of the cadmium enzymes vs. native [(LADH)Zn2Zn2] reveal maxima at 240 nm with molar absorptivities, delta epsilon 240, of 1.6 X 10(4) M-1 cm-1 per noncatalytic 109Cd atom and 0.9 X 10(4) M-1 cm-1 per catalytic 109Cd atom, consistent with coordination of the metals by four and two thiolate ligands, respectively, strikingly similar to the 250-nm charge-transfer absorbance in metallothionein. Carboxymethylation of the Cys-46 ligand to the catalytic metal in LADH presumably lowers the overall stability constant of the coordination complex and results in loss of catalytic 109Cd or catalytic cobalt but not catalytic zinc from the enzyme.     

Accurate sequence-based prediction of catalytic residues

MOTIVATION: Prediction of catalytic residues provides useful information for the research on function of enzymes. Most of the existing prediction methods are based on structural information, which limits their use. We propose a sequence-based catalytic residue predictor that provides predictions with quality comparable to modern structure-based methods and that exceeds quality of state-of-the-art sequence-based methods. RESULTS: Our method (CRpred) uses sequence-based features and the sequence-derived PSI-BLAST profile. We used feature selection to reduce the dimensionality of the input (and explain the input) to support vector machine (SVM) classifier that provides predictions. Tests on eight datasets and side-by-side comparison with six modern structure- and sequence-based predictors show that CRpred provides predictions with quality comparable to current structure-based methods and better than sequence-based methods. The proposed method obtains 15-19% precision and 48-58% TP (true positive) rate, depending on the dataset used. CRpred also provides confidence values that allow selecting a subset of predictions with higher precision. The improved quality is due to newly designed features and careful parameterization of the SVM. The features incorporate amino acids characterized by the highest and the lowest propensities to constitute catalytic residues, Gly that provides flexibility for catalytic sites and sequence motifs characteristic to certain catalytic reactions. Our features indicate that catalytic residues are on average more conserved when compared with the general population of residues and that highly conserved amino acids characterized by high catalytic propensity are likely to form catalytic sites. We also show that local (with respect to the sequence) hydrophobicity contributes towards the prediction.