Catalytic Mechanism Of Enzymes Research Articles

ChemInform Abstract: Repair of 8‐Oxoguanine in DNA. The Mechanisms of Enzymatic Catalysis

AbstractChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 200 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.

Mining for new enzymes

Mining for new enzymes

Force-Clamp Spectroscopy Detects Residue Co-evolution in Enzyme Catalysis

Understanding how the catalytic mechanisms of enzymes are optimized through evolution remains a major challenge in molecular biology. The concept of co-evolution implicates that compensatory mutations occur to preserve the structure and function of proteins. We have combined statistical analysis of protein sequences with the sensitivity of single molecule force-clamp spectroscopy to probe how catalysis is affected by structurally distant correlated mutations in Escherichia coli thioredoxin. Our findings show that evolutionary anti-correlated mutations have an inhibitory effect on enzyme catalysis, whereas positively correlated mutations rescue the catalytic activity. We interpret these results in terms of an evolutionary tuning of both the enzyme-substrate binding process and the chemistry of the active site. Our results constitute a direct observation of distant residue co-evolution in enzyme catalysis.

Modeling catalytic mechanism of nitrile hydratase by semi-empirical quantum mechanical calculation

Nitrile hydratase (NHase) is an important industrial enzyme capable of converting nitriles to corresponding amides. Utilizing the method of semi-empirical quantum mechanical (QM) calculation by TRITON, the bioconversion process of acrylonitrile to acrylamide catalyzed by NHase was successfully performed on a computer. Crystal structure of a Co-type NHase from Pseudonocardia thermophila JCM 3095 (PDB code 1IRE) was selected as the target for acrylonitrile autodock. In silico calculations were performed on the NHase–acrylonitrile complex to simulate the enzyme catalysis mechanism by quantitatively comparing energy changes of each reaction pathway. Simulation results showed that active site activation is the first step of NHase catalysis, in which the Co 2+ coordinated to a water molecule forms a Co–OH complex mediated by the oxidized α-CEA113. Then the oxygen atom in the Co–OH attacks the C atom in the –CN triple bond of acrylonitrile, forming a precursor of acrylamide. Consequently, proton rearrangement happens transforming the precursor into the final product of acrylamide, under the assistance of the hydrogen atom in the –OH group of α-SER112. Gibbs energy changes of three steps corresponding to the active center activation, nucleophilic attack and proton rearrangement are around −31, 23 and −12 kcal/mol, respectively.

An enzyme-centric approach for modelling non-linear biological complexity

BackgroundThe current challenge of Systems Biology is to integrate high throughput data sets for simulating the complexity of biological networks, exploit the evolution of nature-designed networks that maintain the robustness of a biological system, and thereby generate novel, experimentally testable hypotheses. In order to simulate non-linear biological complexities, we have previously developed an Enzyme-Centric mechanistic modeling approach and validated it using metabolic network in E. coli. The idea is to use prior knowledge of catalytic and regulatory mechanisms of each enzyme within the metabolic network to build a dynamic model for investigating the network level regulation and thus understand the nature design principle behind the network.ResultsIn this paper, we further demonstrate the application of complex enzyme catalytic and regulatory modules to simulate nonlinear network regulatory patterns vs. simple linear conversion model. We learned and validated that it is essential to incorporate prior knowledge from the literature to simulate non-linear biological complexities. The network expandability is demonstrated and validated with the complex amino acid biosynthetic network with multi-regulations. Also, we demonstrated the compatibility of mechanistic models within close species. Furthermore, the eukaryotic protein factory model for insuring steady mRNA production is simulated and the coupling of RNA transcription and splicing is validated by both mathematical simulation and experimental analysis.ConclusionWe demonstrated the importance of modeling complex enzyme catalytic and regulatory mechanisms to further understand nonlinear network regulatory patterns. The simulations presented in this paper reveal how a living system maintains homeostasis and its robustness to continue functioning while facing environmental stresses or genetic mutations.

Crystal structure of human phosphomavelonate kinase at 1.8 Å resolution

Crystal structure of human phosphomavelonate kinase at 1.8 Å resolution

Pressure effects on catalytic properties and structural stability of human paraoxonase

Human paraoxonase (HuPON1) is a candidate as catalytic bioscavenger for pre-treatment and therapy of poisoning by organophosphate compounds. HuPON1 is a hydrophobic protein associated with a partner, the human phosphate binding protein (HPBP) in plasma high density lipoproteins. The relationship between the composition and the size of multimeric states of HuPON1 is not well understood. Moreover the effect of HPBP's presence on enzyme catalytic mechanisms and stability is unclear. We investigated the effect of hydrostatic pressure and temperature on structural stability and activity of different PON1 preparations (hybrid recombinant PON1, natural HuPON1 free of its partner or in the presence of 50% w/w HPBP). We showed that PON1 exists under several multimeric forms and that the binding of HPBP amends the size of the hetero-oligomeric states and exerts a stabilizing effect on the activity of PON1.

Implications of a Carboxylate‐Bound C‐Cluster Structure of Carbon Monoxide Dehydrogenase

Seeing the C: Nickel-containing carbon monoxide dehydrogenases reversibly oxidize CO to CO2 at a {[Fe3S4]:[Ni Fea]} active site known as the C-cluster. Recently reported structures of the enzyme by Jeoung and Dobbek, including those of CO2-bound and OH-bound intermediates, shed new light on the enzyme's catalytic mechanism. This Highlight describes these developments and their implications.

Computational enzymology: insight into biological catalysts from modelling

Molecular modelling and simulation can give atomic-level understanding of the fundamental mechanisms of enzyme catalysis. For example, modelling can identify likely enzyme reaction mechanisms, analyse catalytic interactions, and identify determinants of reactivity and specificity. Combined quantum mechanics/molecular mechanics (QM/MM) methods are an important technique in this maturing field of computational enzymology. By coupling quantum chemical (electronic structure) calculations on the active site with a simpler, empirical 'molecular mechanics' treatment of the rest of the protein, QM/MM methods allow the modelling of reactions in enzymes. In this Highlight, QM/MM techniques are outlined and some recent applications are discussed. These applications illustrate how calculations of this type can be used to interpret and complement experiments, with important potential implications for practical developments such as drug and catalyst design.

Structural and Initial Biological Analysis of Synthetic Arylomycin A2

The growing threat of untreatable bacterial infections has refocused efforts to identify new antibiotics, especially those acting by novel mechanisms. While the inhibition of pathogen proteases has proven to be a successful strategy for drug development, such inhibitors are often limited by toxicity due to their promiscuous inhibition of homologous and mechanistically related human enzymes. Unlike many protease inhibitors, inhibitors of the essential type I bacterial signal peptidase (SPase) may be more specific and thus less toxic due to the enzyme's unique structure and catalytic mechanism. Recently, the arylomycins and related lipoglycopeptide natural products were isolated and shown to inhibit SPase. The core structure of the arylomycins and lipoglycopeptides consists of a biaryl-linked, N-methylated peptide macrocycle attached to a lipopeptide tail, and in the case of the lipoglycopeptides, a deoxymannose moiety. Herein, we report the first total synthesis of a member of this group of antibiotics, arylomycin A2. The synthesis relies on Suzuki-Miyaura-mediated biaryl coupling, which model studies suggested would be more efficient than a lactamization-based route. Biological studies demonstrate that these compounds are promising antibiotics, especially against Gram-positive pathogens, with activity against S. epidermidis that equals that of the currently prescribed antibiotics. Structural and biological studies suggest that both N-methylation and lipidation may contribute to antibiotic activity, whereas glycosylation appears to be generally less critical. Thus, these studies help identify the determinants of the biological activity of arylomycin A2 and should aid in the design of analogs to further explore and develop this novel class of antibiotic.

The Crystal Structures of Ornithine Carbamoyltransferase from Mycobacterium tuberculosis and Its Ternary Complex with Carbamoyl Phosphate and l-Norvaline Reveal the Enzyme's Catalytic Mechanism

Mycobacterium tuberculosis ornithine carbamoyltransferase ( Mtb OTC) catalyzes the sixth step in arginine biosynthesis; it produces citrulline from carbamoyl phosphate (CP) and ornithine (ORN). Here, we report the crystal structures of Mtb OTC in orthorhombic (form I) and hexagonal (form II) space groups. The molecules in form II are complexed with CP and l-norvaline (NVA); the latter is a competitive inhibitor of OTC. The asymmetric unit in form I contains a pseudo hexamer with 32 point group symmetry. The CP and NVA in form II induce a remarkable conformational change in the 80s and the 240s loops with the displacement of these loops towards the active site. The displacement of these loops is strikingly different from that seen in other OTC structures. In addition, the ligands induce a domain closure of 4.4° in form II. Sequence comparison of active-site residues of Mtb OTC with several other OTCs of known structure reveals that they are virtually identical. The interactions involving the active-site residues of Mtb OTC with CP and NVA and a modeling study of ORN in the form II structure strongly rule out an earlier proposed mechanistic role of Cys264 in catalysis and suggest a possible mechanism for OTC. Our results strongly support the view that ORN with an already deprotonated N ε atom is the species that binds to the enzyme and that one of the phosphate oxygen atoms of CP is likely to be involved in accepting a proton from the doubly protonated N ε atom of ORN. We have interpreted this deprotonation as part of the collapse of the transition state of the reaction.

Mechanisms of enzyme catalysis from crystal structure analyses.

The enzyme families to which glutathione reductase and adenylate kinase belong show appreciably different structural behaviour during catalysis. While the electron transfer in glutathione reductase requires only small movements of a couple of non-hydrogen atoms, the transfer of a phosphoryl group in the nucleotide kinases involves major rearrangements with main chain displacements of up to 32 A. In both cases, the structural changes during a catalytic cycle have been derived from a number of separate X-ray crystallographic analyses.

The Chemistry of Protein Catalysis

We report, for the first time, on the statistics of chemical mechanisms and amino acid residue functions that occur in enzyme reaction sequences using the MACiE database of 202 distinct enzyme reaction mechanisms as a knowledge base. MACiE currently holds representatives from each Enzyme Commission sub-subclass where there is an available crystal structure and sufficient evidence in the primary literature for a mechanism. Each catalytic step of every reaction sequence in MACiE is fully annotated, so that it includes the function of the catalytic residues involved in the reaction and the chemical mechanisms by which substrates are transformed into products. We show that the most catalytic amino acid residues are histidine, cysteine and aspartate, which are also the residues whose side-chains are more likely to serve as reactants, and that have the greatest versatility of function. We show that electrophilic reactions in enzymes are very rare, and the majority of enzyme reactions rely upon nucleophilic and general acid/base chemistry. However, although rare, radical (homolytic) reactions are much more common than electrophilic reactions. Thus, the majority of amino acid residues perform stabilisation roles (as spectators) or proton shuttling roles (as reactants). The analysis presented provides a better understanding of the mechanisms of enzyme catalysis and may act as an initial step in the validation and prediction of mechanism in an enzyme active site.

Proteomic analysis of N‐glycosylation in mosquito dopachrome conversion enzyme

A novel dopachrome conversion enzyme (DCE) is present in insects and involved in their melanization pathway. DCE shares no sequence homology with any noninsect species from bacteria to humans. Several DCE sequences have been available, but enzyme structure and catalytic mechanism are unclear. This study concerns DCE PTMs, especially glycosylation. A mosquito DCE was purified and its monosaccharide composition, N-glycosylation site, and oligosaccharide structures were determined. Results showed that N-acetyl D-glucosamine and D-mannose are the major monosaccharides and L-fucose, D-xylose, and D-arabinose are the minor ones in mosquito DCE. Glycosylation site and oligosaccharide structures were elucidated from MS and MS/MS spectra of trypsin-digested DCE glycopeptides. A single N-glycosylation site (Asn285 -Glu-Thr) was identified in DCE and was proven to be fully glycosylated. Man3GlcNAc2, Man3(Fuc)1-2GlcNAc2, and their truncated structures were the dominant oligosaccharides. In addition, high mannose-type structures (Man4-7(Fuc)GlcNAc2) were also identified. Removal of DCE N-oligosaccharides with peptide N-glycosidase (PNGase F) decreased its activity and thermal stability. However, partial DCE deglycosylation with alpha-mannosidase or alpha-fucosidase somewhat stimulated its activity and improved its thermal stability. During mass spectrometric analysis of DCE glycopeptides, their CID patterns were highly intriguing, in that some glycopeptides underwent both C-terminal rearrangement and formation of dimeric structures during CID. Results of this study provide an interesting example in terms of potential complexity of the glycopeptide CID fragmentation pattern.

Structure and Molecular Mechanism of Bacillus anthracis Cofactor-Independent Phosphoglycerate Mutase: A Crucial Enzyme for Spores and Growing Cells of Bacillus Species

Phosphoglycerate mutases (PGMs) catalyze the isomerization of 2- and 3-phosphoglycerates and are essential for glucose metabolism in most organisms. This study reports the production, structure, and molecular dynamics analysis of Bacillus anthracis cofactor-independent PGM (iPGM). The three-dimensional structure of B. anthracis PGM is composed of two structural and functional domains, the phosphatase and transferase. The structural relationship between these two domains is different than in the B. stearothermophilus iPGM structure determined previously. However, the structures of the two domains of B. anthracis iPGM show a high degree of similarity to those in B. stearothermophilus iPGM. The novel domain arrangement in B. anthracis iPGM and the dynamic property of these domains is directly linked to the mechanism of enzyme catalysis, in which substrate binding is proposed to result in close association of the two domains. The structure of B. anthracis iPGM and the molecular dynamics of this structure provide unique insight into the mechanism of iPGM catalysis, in particular the roles of changes in coordination geometry of the enzyme’s two bivalent metal ions and the regulation of this enzyme’s activity by changes in intracellular pH during spore formation and germination in Bacillus species.

2P314 光化学系IIにおける水分解・酸素発生反応の新しい酵素触媒機構(光生物学(光合成・視覚と光受容),口頭発表,第45回日本生物物理学会年会)

2P314 光化学系IIにおける水分解・酸素発生反応の新しい酵素触媒機構(光生物学(光合成・視覚と光受容),口頭発表,第45回日本生物物理学会年会)

Quaternary Structure, Substrate Selectivity and Inhibitor Design for SARS 3C-Like Proteinase

The SARS coronavirus 3C-like proteinase is recognized as a potential drug design target for the treatment of severe acute respiratory syndrome. In the past few years, much work has been done to understand the catalytic mechanism of this target protein and to design its selective inhibitors. The protein exists as a dimer/monomer mixture in solution and the dimer was confirmed to be the active species for the enzyme reaction. Quantitative dissociation constants have been reported for the dimer by using analytic ultracentrifuge, gel filtration and enzyme assays. Though the enzyme is a cysteine protease with a chymotrypsin fold, SARS 3C-like proteinase follows the general base catalytic mechanism similar to chymotrypsin. As the enzyme can cut eleven different sites on the viral polyprotein, the substrate specificity has been studied by synthesized peptides corresponding or similar to the cleavage sites on the polyprotein. Predictive model was built for substrate structure and activity relationships and can be applied in inhibitor design. Due to the lack of potential drugs for the treatment of SARS, the discovery of inhibitors against SARS 3C-like proteinase, which can potentially be optimized as drugs appears to be highly desirable. Various groups have been working on inhibitor discovery by virtual screening, compound library screening, modification of existing compounds or natural products. High-throughput in vitro assays, auto-cleavage assays and viral replication assays have been developed for inhibition activity tests. Inhibitors with IC50 values as low as 60 nM have been reported.

Sequence analysis and structure prediction of type II Pseudomonas sp. USM 4–55 PHA synthase and an insight into its catalytic mechanism

BackgroundPolyhydroxyalkanoates (PHA), are biodegradable polyesters derived from many microorganisms such as the pseudomonads. These polyesters are in great demand especially in the packaging industries, the medical line as well as the paint industries. The enzyme responsible in catalyzing the formation of PHA is PHA synthase. Due to the limited structural information, its functional properties including catalysis are lacking. Therefore, this study seeks to investigate the structural properties as well as its catalytic mechanism by predicting the three-dimensional (3D) model of the Type II Pseudomonas sp. USM 4–55 PHA synthase 1 (PhaC1P.sp USM 4–55).ResultsSequence analysis demonstrated that PhaC1P.sp USM 4–55 lacked similarity with all known structures in databases. PSI-BLAST and HMM Superfamily analyses demonstrated that this enzyme belongs to the alpha/beta hydrolase fold family. Threading approach revealed that the most suitable template to use was the human gastric lipase (PDB ID: 1HLG). The superimposition of the predicted PhaC1P.sp USM 4–55 model with 1HLG covering 86.2% of the backbone atoms showed an RMSD of 1.15 Å. The catalytic residues comprising of Cys296, Asp451 and His479 were found to be conserved and located adjacent to each other. In addition to this, an extension to the catalytic mechanism was also proposed whereby two tetrahedral intermediates were believed to form during the PHA biosynthesis. These transition state intermediates were further postulated to be stabilized by the formation of oxyanion holes. Based on the sequence analysis and the deduced model, Ser297 was postulated to contribute to the formation of the oxyanion hole.ConclusionThe 3D model of the core region of PhaC1P.sp USM 4–55 from residue 267 to residue 484 was developed using computational techniques and the locations of the catalytic residues were identified. Results from this study for the first time highlighted Ser297 potentially playing an important role in the enzyme's catalytic mechanism.

The N-terminal octapeptide acts as a dimerization inhibitor of SARS coronavirus 3C-like proteinase

The 3C-like proteinase of severe acute respiratory syndrome (SARS) coronavirus has been proposed to be a key target for structural-based drug design against SARS. Accurate determination of the dimer dissociation constant and the role of the N-finger (residues 1–7) will provide more insights into the enzyme catalytic mechanism of SARS 3CL proteinase. The dimer dissociation constant of the wild-type protein was determined to be 14.0 μM by analytical ultracentrifugation method. The N-finger fragment of the enzyme plays an important role in enzyme dimerization as shown in the crystal structure. Key residues in the N-finger have been studied by site-directed mutagenesis, enzyme assay, and analytical ultracentrifugation. A single mutation of M6A was found to be critical to maintain the dimer structure of the enzyme. The N-terminal octapeptide N8 and its mutants were also synthesized and tested for their potency as dimerization inhibitors. Peptide cleavage assay confirms that peptide N8 is a dimerization inhibitor with a K i of 2.20 mM. The comparison of the inhibitory activities of N8 and its mutants indicates that the hydrophobic interaction of Met-6 and the electrostatic interaction of Arg-4 contribute most for inhibitor binding. This study describes the first example of inhibitors targeting the dimeric interface of SARS 3CL proteinase, providing a novel strategy for drug design against SARS and other coronaviruses.

MACiE: a database of enzyme reaction mechanisms

MACiE (mechanism, annotation and classification in enzymes) is a publicly available web-based database, held in CMLReact (an XML application), that aims to help our understanding of the evolution of enzyme catalytic mechanisms and also to create a classification system which reflects the actual chemical mechanism (catalytic steps) of an enzyme reaction, not only the overall reaction. http://www-mitchell.ch.cam.ac.uk/macie/.