Binding Affinity Of Enzyme Research Articles

Binding affinity and adhesion force of organophosphate hydrolase enzyme with soil particles related to the isoelectric point of the enzyme

The binding affinity of organophosphate hydrolase enzyme (OphB) with soil particles in relation to the isoelectric point (pI) was studied. Immobilization of OphB with soil particles was observed by confocal microscopy, Fourier transform infrared spectroscopy (FT-IR), and Atomic force microscopy (AFM). The calculated pI of OphB enzyme was increased from 8.69 to 8.89, 9.04 and 9.16 by the single, double and triple mutant of OphB enzyme, respectively through the replacement of negatively charged aspartate with positively charged histidine. Practically, the binding affinity was increased to 5.30%, 11.50%, and 16.80% for single, double and triple mutants, respectively. In contrast, enzyme activity of OphB did not change by the mutation of the enzyme. On the other hand, adhesion forces were gradually increased for wild type OphB enzyme (90 pN) to 96, 100 and 104 pN for single, double and triple mutants of OphB enzyme, respectively. There was an increasing trend of binding affinity and adhesion force by the increase of isoelectric point (pI) of OphB enzyme.

PH-Responsive Pharmacological Chaperones for Rescuing Mutant Glycosidases.

A general approach is reported for the design of small-molecule competitive inhibitors of lysosomal glycosidases programmed to 1) promote correct folding of mutant enzymes at the endoplasmic reticulum, 2) facilitate trafficking, and 3) undergo dissociation and self-inactivation at the lysosome. The strategy is based on the incorporation of an orthoester segment into iminosugar conjugates to switch the nature of the aglycone moiety from hydrophobic to hydrophilic in the pH 7 to pH 5 window, which has a dramatic effect on the enzyme binding affinity. As a proof of concept, new highly pH-responsive glycomimetics targeting human glucocerebrosidase or α-galactosidase with strong potential as pharmacological chaperones for Gaucher or Fabry disease, respectively, were developed.

PH‐Responsive Pharmacological Chaperones for Rescuing Mutant Glycosidases

AbstractA general approach is reported for the design of small‐molecule competitive inhibitors of lysosomal glycosidases programmed to 1) promote correct folding of mutant enzymes at the endoplasmic reticulum, 2) facilitate trafficking, and 3) undergo dissociation and self‐inactivation at the lysosome. The strategy is based on the incorporation of an orthoester segment into iminosugar conjugates to switch the nature of the aglycone moiety from hydrophobic to hydrophilic in the pH 7 to pH 5 window, which has a dramatic effect on the enzyme binding affinity. As a proof of concept, new highly pH‐responsive glycomimetics targeting human glucocerebrosidase or α‐galactosidase with strong potential as pharmacological chaperones for Gaucher or Fabry disease, respectively, were developed.

Partially reduced graphene oxide–gold nanorods composite based bioelectrode of improved sensing performance

The present work proposes partially reduced graphene oxide–gold nanorods supported by chitosan (CH–prGO–AuNRs) as a potential bioelectrode material for enhanced glucose sensing. Developed on ITO substrate by immobilizing glucose oxidase on CH–prGO–AuNRs composite, these CH–prGO–AuNRs/ITO bioelectrodes demonstrate high sensitivity of 3.2µA/(mg/dL)/cm2 and linear range of 25–200mg/dL with an ability to detect as low as 14.5mg/dL. Further, these CH–prGO–AuNRs/ITO based electrodes attest synergistiacally enhanced sensing properties when compared to simple graphene oxide based CH–GO/ITO electrode. This is evident from one order higher electron transfer rate constant (Ks) value in case of CH–prGO–AuNRs modified electrode (12.4×10–2cm/s), in contrast to CH–GO/ITO electrode (6×10–3cm/s). Additionally, very low Km value [15.4mg/dL(0.85mM)] ensures better binding affinity of enzyme to substrate which is desirable for good biosensor stability and resistance to environmental interferences. Hence, with better loading capacity, kinetics and stability, the proposed CH–prGO–AuNRs composite shows tremendous potential to detect several bio-analytes in the coming future.

TRMT10A dysfunction is associated with abnormalities in glucose homeostasis, short stature and microcephaly

BackgroundTrm10 is a tRNA m1G9 methyltransferase, which in yeast modifies 12 different tRNA species, yet is considered non-essential for viability under standard growth conditions. In humans, there are three Trm10...

Engineered cytidine triphosphate synthetase with reduced product inhibition.

Cytidine triphosphate (CTP) synthetase (CTPS) (EC number 6.3.4.2) is a key enzyme involved in de novo synthesis of CTP. It catalyzes the rate-limiting step of the process due to the product inhibition effects on the enzyme. In this study, a novel CTPS from Corynebacterium glutamicum ATCC 13032 (CgCTPS) was cloned, expressed and characterized. A series of mutagenesis in its N-terminal ammonia ligase (ALase) domain was performed in order to reduce CTP product inhibition. All single mutation variants (D160E, E162A, E168K) lowered product inhibition by lowering the enzyme's binding affinity for CTP. The homology model of CgCTPS showed that D160E mutant caused steric hindrance for the pyrimidine ring of CTP stacking, E162A disrupted the hydrogen bond between CTP ribose and side chain and D168K caused minor localized structure perturbations of CTP binding pocket. The triple mutant of CTPS (D160E-E162A-E168K) with halved Km, doubled Vmax and the 23.5-fold increased IC50 for CTP shows a potential for use in industrial-scale CTP production by its better performance in enzyme kinetics and product inhibition.

Rapid Identification of Cholinesterase Inhibitors from the Seedcases of Mangosteen Using an Enzyme Affinity Assay

Enzyme binding affinity has been recently introduced as a selective screening method to identify bioactive substances within complex mixtures. We used an assay which identified small molecule binders of acetylcholinesterase (AChE) using the following series of steps: incubation of enzyme with extract; centrifugation and filtration; identification of small molecule content in the flow through. The crude extract contained 10 peaks in the UPLC chromatogram. However, after incubation the enzyme, six peaks were reduced, indicating these compounds bound AChE. All these isolated compounds (2, 3, and 5-8) significantly inhibited human AChE with IC₅₀s = 5.4-15.0 μM and butyrylcholinsterase (IC₅₀s = 0.7-11.0 μM). All compounds exhibited reversible mixed kinetics. Consistent with the binding screen and fluorescence quenching, γ-mangostin 6 had a much higher affinity for AChE than 9-hydroxycalabaxanthone 9. This validates this screening protocol as a rapid method to identify inhibitors of AChE.

Nature's inordinate fondness for metabolic enzymes: why metabolic enzyme loci are so frequently targets of selection

Metabolic enzyme loci were some of the first genes accessible for molecular evolution and ecology research. New technologies now make the whole genome, transcriptome or proteome readily accessible, allowing unbiased scans for loci exhibiting significant differences in allele frequency or expression level and associated with phenotypes and/or responses to natural selection. With surprising frequency and in many cases in proportions greater than chance relative to other genes, glycolysis and TCA cycle enzyme loci appear among the genes with significant associations in these studies. Hence, there is an ongoing need to understand the basis for fitness effects of metabolic enzyme polymorphisms. Allele-specific effects on the binding affinity and catalytic rate of individual enzymes are well known, but often of uncertain significance because metabolic control theory and in vivo studies indicate that many individual metabolic enzymes do not affect pathway flux rate. I review research, so far little used in evolutionary biology, showing that metabolic enzyme substrates affect signalling pathways that regulate cell and organismal biology, and that these enzymes have moonlighting functions. To date there is little knowledge of how alleles in natural populations affect these phenotypes. I discuss an example in which alleles of a TCA enzyme locus associate with differences in a signalling pathway and development, organismal performance, and ecological dynamics. Ultimately, understanding how metabolic enzyme polymorphisms map to phenotypes and fitness remains a compelling and ongoing need for gaining robust knowledge of ecological and evolutionary processes.

Prohormone-substrate peptide sequence recognition by peptidylglycine α-amidating monooxygenase and its reflection in increased glycolate inhibitor potency

The interactions of nineteen peptide substrates and fifteen analogous peptidomimetic glycolate inhibitors with human peptidylglycine α-amidating monooxygenase (PAM) have been investigated. The substrates and inhibitors are the prohormones of calcitonin and oxytocin and their analogues. PAM both secreted into the medium by and extracted from DMS53 small lung carcinoma cells has been studied. The results show that recognition of the prooxytocin and procalcitonin peptide sequences by the enzyme extends more than four and five amino acid residues, respectively, from their C-termini. This substrate sequence recognition is mirrored by increased inhibitor potency with increased peptide length in the glycolate peptidomimetics. Substitution of the C-terminal penultimate glycine and proline residues of prooxytocin and procalcitonin and their analogues with phenylalanine increases the enzyme binding affinity. However, this changes the binding mode from one that depends on peptide sequence recognition to another primarily determined by the phenylalanine moiety, for both the substrates and analogous glycolate inhibitors.

Control of Enzyme–Solid Interactions via Chemical Modification

Electrostatic forces could contribute significantly toward enzyme-solid interactions, and controlling these charge-charge interactions while maintaining high affinity, benign adsorption of enzymes on solids is a challenge. Here, we demonstrate that chemical modification of the surface carboxyl groups of enzymes can be used to adjust the net charge of the enzyme and control binding affinities to solid surfaces. Negatively charged nanosolid, α-Zr(HPO(4))(2)·H(2)O (abbreviated as α-ZrP) and two negatively charged proteins, glucose oxidase (GO) and methemoglobin (Hb), have been chosen as model systems. A limited number of the aspartate and glutamate side chains of these proteins are covalently modified with tetraethylenepentamine (TEPA) to convert these negatively charged proteins into the corresponding positively charged ones (cationized). Cationized proteins retained their structure and activities to a significant extent, and the influence of cationization on binding affinities has been tested. Cationized GO, for example, showed 250-fold increase in affinity for the negatively charged α-ZrP, when compared to that of the unmodified GO, and cationized Hb, similarly, indicated 26-fold increase in affinity. Circular dichroism spectra showed that α-ZrP-bound cationized GO retained native-like structure to a significant extent, and activity studies showed that cationized GO/α-ZrP complex is ~2.5-fold more active than GO/α-ZrP. Cationized Hb/α-ZrP retained ~75% of activity of Hb/α-ZrP. Therefore, enzyme cationization enhanced affinities by 1-2 orders of magnitude, while retaining considerable activity for the bound biocatalyst. This benign, chemical control over enzyme charge provided a powerful new strategy to rationally modulate enzyme-solid interactions while retaining their biocatalytic properties.

Role of Conserved Glycine in Zinc-dependent Medium Chain Dehydrogenase/Reductase Superfamily

The medium-chain dehydrogenase/reductase (MDR) superfamily consists of a large group of enzymes with a broad range of activities. Members of this superfamily are currently the subject of intensive investigation, but many aspects, including the zinc dependence of MDR superfamily proteins, have not yet have been adequately investigated. Using a density functional theory-based screening strategy, we have identified a strictly conserved glycine residue (Gly) in the zinc-dependent MDR superfamily. To elucidate the role of this conserved Gly in MDR, we carried out a comprehensive structural, functional, and computational analysis of four MDR enzymes through a series of studies including site-directed mutagenesis, isothermal titration calorimetry, electron paramagnetic resonance (EPR), quantum mechanics, and molecular mechanics analysis. Gly substitution by other amino acids posed a significant threat to the metal binding affinity and activity of MDR superfamily enzymes. Mutagenesis at the conserved Gly resulted in alterations in the coordination of the catalytic zinc ion, with concomitant changes in metal-ligand bond length, bond angle, and the affinity (K(d)) toward the zinc ion. The Gly mutants also showed different spectroscopic properties in EPR compared with those of the wild type, indicating that the binding geometries of the zinc to the zinc binding ligands were changed by the mutation. The present results demonstrate that the conserved Gly in the GHE motif plays a role in maintaining the metal binding affinity and the electronic state of the catalytic zinc ion during catalysis of the MDR superfamily enzymes.

Substituted aminopyrimidine protein kinase B (PknB) inhibitors show activity against Mycobacterium tuberculosis

A high-throughput screen against PknB, an essential serine–threonine protein kinase present in Mycobacterium tuberculosis (M. tuberculosis), allowed the identification of an aminoquinazoline inhibitor which was used as a starting point for SAR investigations. Although a significant improvement in enzyme affinity was achieved, the aminoquinazolines showed little or no cellular activity against M. tuberculosis. However, switching to an aminopyrimidine core scaffold and the introduction of a basic amine side chain afforded compounds with nanomolar enzyme binding affinity and micromolar minimum inhibitory concentrations against M. tuberculosis. Replacement of the pyrazole head group with pyridine then allowed equipotent compounds with improved selectivity against a human kinase panel to be obtained.

Manipulation of enzyme properties by noncanonical amino acid incorporation

Since wild-type enzymes do not always have the properties needed for various applications, enzymes are often engineered to obtain desirable properties through protein engineering techniques. In the past decade, complementary to the widely used rational protein design and directed evolution techniques, noncanonical amino acid incorporation (NCAAI) has become a new and effective protein engineering technique. Recently, NCAAI has been used to improve intrinsic functions of proteins, such as enzymes and fluorescent proteins, beyond the capacities obtained with natural amino acids. Herein, recent progress on improving enzyme properties through NCAAI in vivo is reviewed and the challenges of current approaches and future directions are also discussed. To date, both NCAAI methods-residue- and site-specific incorporation-have been primarily used to improve the catalytic turnover number and substrate binding affinity of enzymes. Numerous strategies used to minimize structural perturbation and stability loss of a target enzyme upon NCAAI are also explored. Considering the generality of NCAAI incorporation, we expect its application could be expanded to improve other enzyme properties, such as substrate specificity and solvent resistance in the near future.

Structural prediction and comparative docking studies of psychrophilic β- Galactosidase with lactose, ONPG and PNPG against its counter parts of mesophilic and thermophilic enzymes.

Enzymes from psychrophiles catalyze the reactions at low temperatures with higher specific activity. Among all the psychrophilic enzymes produced, cold active β-galactosidase from marine psychrophiles revalorizes a new arena in numerous areas at industrial level. The hydrolysis of lactose in to glucose and galactose by cold active β-galactosidase offers a new promising approach in removal of lactose from milk to overcome the problem of lactose intolerance. Herein we propose, a 3D structure of cold active β-galactosidase enzyme sourced from Pseudoalteromonas haloplanktis by using Modeler 9v8 and best model was developed having 88% of favourable region in ramachandran plot. Modelling was followed by docking studies with the help of Auto dock 4.0 against the three substrates lactose, ONPG and PNPG. In addition, comparative docking studies were also performed for the 3D model of psychrophilic β-galactosidase with mesophilic and thermophilic enzymes. Docking studies revealed that binding affinity of enzyme towards the three different substrates is more for psychrophilic enzyme when compared with mesophilic and thermophilic enzymes. It indicates that the enzyme has high specific activity at low temperature when compared with mesophilic and thermophilic enzymes.

Accelerated Search Kinetics Mediated by Redox Reactions of DNA Repair Enzymes

A charge transport (CT) mechanism has been proposed in several articles to explain the localization of base excision repair (BER) enzymes to lesions on DNA. The CT mechanism relies on redox reactions of iron-sulfur cofactors that modify the enzyme's binding affinity. These redox reactions are mediated by the DNA strand and involve the exchange of electrons between BER enzymes along DNA. We propose a mathematical model that incorporates enzyme binding/unbinding, electron transport, and enzyme diffusion along DNA. Analysis of our model within a range of parameter values suggests that the redox reactions can increase desorption of BER enzymes not already bound to lesions, allowing the enzymes to be recycled—thus accelerating the overall search process. This acceleration mechanism is most effective when enzyme copy numbers and enzyme diffusivity along the DNA are small. Under such conditions, we find that CT BER enzymes find their targets more quickly than simple passive enzymes that simply attach to the DNA without desorbing.

Structural comparison of chromosomal and exogenous dihydrofolate reductase from Staphylococcus aureus in complex with the potent inhibitor trimethoprim

Dihydrofolate reductase (DHFR) is the enzyme responsible for the NADPH-dependent reduction of 5,6-dihydrofolate to 5,6,7,8-tetrahydrofolate, an essential cofactor in the synthesis of purines, thymidylate, methionine, and other key metabolites. Because of its importance in multiple cellular functions, DHFR has been the subject of much research targeting the enzyme with anticancer, antibacterial, and antimicrobial agents. Clinically used compounds targeting DHFR include methotrexate for the treatment of cancer and diaminopyrimidines (DAPs) such as trimethoprim (TMP) for the treatment of bacterial infections. DAP inhibitors of DHFR have been used clinically for >30 years and resistance to these agents has become widespread. Methicillin-resistant Staphylococcus aureus (MRSA), the causative agent of many serious nosocomial and community acquired infections, and other gram-positive organisms can show resistance to DAPs through mutation of the chromosomal gene or acquisition of an alternative DHFR termed "S1 DHFR." To develop new therapies for health threats such as MRSA, it is important to understand the molecular basis of DAP resistance. Here, we report the crystal structure of the wild-type chromosomal DHFR from S. aureus in complex with NADPH and TMP. We have also solved the structure of the exogenous, TMP resistant S1 DHFR, apo and in complex with TMP. The structural and thermodynamic data point to important molecular differences between the two enzymes that lead to dramatically reduced affinity of DAPs to S1 DHFR. These differences in enzyme binding affinity translate into reduced antibacterial activity against strains of S. aureus that express S1 DHFR.

PCNA stimulates catalysis by structure-specific nucleases using two distinct mechanisms: substrate targeting and catalytic step

The sliding clamp Proliferating Cell Nuclear Antigen (PCNA) functions as a recruiter and organizer of a wide variety of DNA modifying enzymes including nucleases, helicases, polymerases and glycosylases. The 5′-flap endonuclease Fen-1 is essential for Okazaki fragment processing in eukaryotes and archaea, and is targeted to the replication fork by PCNA. Crenarchaeal XPF, a 3′-flap endonuclease, is also stimulated by PCNA in vitro. Using a novel continuous fluorimetric assay, we demonstrate that PCNA activates these two nucleases by fundamentally different mechanisms. PCNA stimulates Fen-1 by increasing the enzyme's binding affinity for substrates, as suggested previously. However, PCNA activates XPF by increasing the catalytic rate constant by four orders of magnitude without affecting the KM. PCNA may function as a platform upon which XPF exerts force to distort DNA substrates, destabilizing the substrate and/or stabilizing the transition state structure. This suggests that PCNA can function directly in supporting catalysis as an essential cofactor in some circumstances, a new role for a protein that is generally assumed to perform a passive targeting and organizing function in molecular biology. This could provide a mechanism for the exquisite control of nuclease activity targeted to specific circumstances, such as replication forks or damaged DNA with pre-loaded PCNA.

Synthesis and Conformational Analysis of Stevastelin C3 Analogues and Their Activity Against the Dual‐Specific Vaccina H1‐Related Phosphatase

The biological activity of macrocyclic natural products depends on their conformational properties. For both the elucidation of enzyme binding affinities as well as the development of selective drugs, rigid macrocyclic scaffolds carry high potential. In this study, 13-membered cyclodepsipeptides based on the structure of naturally occurring stevastelins were studied in detail. Six diastereomeric stevastelin C3 analogues and four phosphorylated derivatives were synthesized. The synthesis of linear precursors was achieved on solid support by starting from stereoisomerically pure 2-methyl-3-hydroxy acids. Subsequent macro-lactamization gave the cyclic depsipeptides in very good yields (36-62%). The conformational space of these stevastelin C3 analogues was computationally investigated. On the basis of NMR spectroscopic data, homogeneous conformations were determined for each benzylated depsipeptide and the influence of phosphorylation on the overall conformation was investigated. Importantly, phosphorylation was found to significantly weaken the conformational preferences of the 13-membered depsipeptides. Finally, the cyclic depsipeptides were tested for activity against phosphatases. Inhibitory activity on vaccina H1-related phosphatase was observed depending on the derivatization of the cycles. The activity profiles are discussed in the light of the structural data.

Residues Asp164 and Glu165 at the substrate entryway function potently in substrate orientation of alanine racemase from E. coli: Enzymatic characterization with crystal structure analysis

Alanine racemase (Alr) is an important enzyme that catalyzes the interconversion of L-alanine and D-alanine, an essential building block in the peptidoglycan biosynthesis. For the small size of the Alr active site, its conserved substrate entryway has been proposed as a potential choice for drug design. In this work, we fully analyzed the crystal structures of the native, the D-cycloserine-bound, and four mutants (P219A, E221A, E221K, and E221P) of biosynthetic Alr from Escherichia coli (EcAlr) and studied the potential roles in substrate orientation for the key residues involved in the substrate entryway in conjunction with the enzymatic assays. Structurally, it was discovered that EcAlr is similar to the Pseudomonas aeruginosa catabolic Alr in both overall and active site geometries. Mutation of the conserved negatively charged residue aspartate 164 or glutamate 165 at the substrate entryway could obviously reduce the binding affinity of enzyme against the substrate and decrease the turnover numbers in both D- to L-Ala and L- to D-Ala directions, especially when mutated to lysine with the opposite charge. However, mutation of Pro219 or Glu221 had only negligible or a small influence on the enzymatic activity. Together with the enzymatic and structural investigation results, we thus proposed that the negatively charged residues Asp164 and Glu165 around the substrate entryway play an important role in substrate orientation with cooperation of the positively charged Arg280 and Arg300 on the opposite monomer. Our findings are expected to provide some useful structural information for inhibitor design targeting the substrate entryway of Alr.

CDNA Cloning, Protein Structure Modeling of Rhesus Monkey ( Macaca mulatta) Prothrombin

Objective Hemorrhagic diseases have been considered to be one of the main causes of xenotransplantation failure. To explore the role of the rhesus monkey ( Macaca mulatta) coagulation system in a pig-to-human xenotransplantation model, we primarily studied the full-length cDNA and the three-dimensional (3-D) structures of the important coagulation factor-prothrombin of the rhesus. Materials and Methods The full-length cDNA of rhesus prothrombin was obtained by rhesus monkey liver cDNA library screening and a 5′RACE technique. The 3-D protein structure was modeled using the SWISS-MODEL program. The important macromolecular interaction sites were compared with human and porcine prothrombin. Results At first, the full-length rhesus prothrombin cDNA was cloned; the sequence was submitted to the Genebank (accession number: EF057490). The full-length cDNA is 2029 bp, with a complete open reading frame of 1884 bp, coding 626 amino acids. The deduced protein sequence contains a signal peptide, propeptide, Gla domain, two Kringle domains, and a Trypsin domain. The nucleotide similarities of rhesus-human and rhesus-porcine genes are 95.64% and 86.14%, and those of the amino acids, 94.51% and 82.82%, respectively. Some important functional sites, such as the catalytic triad DHS, RGD, Na +-binding, and the carboxylase-binding site, are highly conserved. However, among the three species some variations are observed in potential N-glycosylation, O-glycosylation, phosphorylation, cleavage site, FXa-binding sites. Especially, the autolysis loop shows great differences in both amino acid sequence and 3-D model. Conclusions The great similarity of prothrombin among rhesus, human, and porcine confirmed the great value of the pig-to-rhesus xenotransplantation model. The variation especially in some important recognition sites between rhesus and pig would vary the binding affinity of enzymes to xenosubstrates and their reaction velocities in postoperative coagulation processes, which would be one possible reason for the disordered regulation of clotting seen during the xenorejection in animal models.