ADP Binding Sites Research Articles

Analyzing Interaction of Rhodacyanine Inhibitor 'MKT-077' with Plasmodium falciparum HSP70s.

MKT-077 and its derivatives are rhodacyanine inhibitors that hold potential in the treatment of cancer, neurodegenerative diseases and malaria. These allosteric drugs act by inhibiting the ATPase action of heat shock proteins of 70 kDa (HSP70). MKT-077 accumulates in the mitochondria and displays differential activity against HSP70 homologs. The four Plasmodium falciparum HSP70s (PfHSP70) are present in various subcellular locations to perform distinct functions. In the present study, we have used bioinformatics tools to understand the interaction of MKT-077 at the ADP and HEW (2-amino 4 bromopyridine) binding sites on PfHSP70s. Our molecular docking experiments predict that the mitochondrial and endoplasmic reticulum PfHSP70 homologs are likely to bind MKT-077 with higher affinities at their ADP binding sites. Binding analysis indicates that the nature of the identified interactions is primarily hydrophobic. We have also identified specific residues of PfHSP70s that are involved in interacting with the ligand. Information obtained in this study may form the foundation for the design and development of MKT-077-based drugs against malaria.

Metabolic Profiling of Mimusops elengi Linn. Leaves extract and in silico anti-inflammatory assessment targeting NLRP3 inflammasome

Mimusops elengi Linn. Secondary metabolites of flavonoids, phenolic acids, coumarin classes and stilbene were identified by UPLC/ESI-QTOF-HRMS/MS technique with negative ion detection. Major Mimusops elengi flavonoids included Myricitrin, Myricetin, and Kaempferol-3-O-alpha-L-rhamnoside. The most abundant Coumarin and phenolic acids detected in the chromatogram included aesculin and quinic acid respectively. Down regulation of NLRP3 inflammasome activation inhibits the severe inflammatory responses caused by virus infection. Studying in silicobinding affinity of flavonoids, coumarins and phenolic acid in M. elengi leaves extract against the ADP binding site of NLRP3 protein (PDB code: 6NPY) demonstrated that investigated compounds have docking scores ranged from −6.20 to −12.30 kcal/mol. The best score was achieved by kaempferol-3-O-(6-p-coumaroyl) -glucoside(Compound 9) followed by aesculin (Compound 25) while Quinic acid (Compound 20) showed the lowest affinity toward ADP-binding site of NLRP3.

Tracking lipid-induced effects on membrane protein structure in real time.

Lipids in biological membranes can act as allosteric modulators of membrane protein function. P-type ATPase proteins carry out ATP-dependent transport of mostly ions across several different types of membranes. It has been shown that both general properties such as membrane thickness and interactions to lipids at specific protein sites dictate P-type ATPase activity. We have developed a methodology to track P-type ATPase domain rearrangements in real time triggered by laser-induced release of ATP and subsequent monitoring by synchrotron X-ray pulses. This time-resolved X-ray solution scattering (TR-XSS) technique was used to capture the dynamics of an equilibrium state of the sarco/endoplasmic reticulum Ca2+ ATPase (SERCA1a), and transient states at 1.5 and 13 milliseconds. The 13-millisecond state showed a previously unresolved actuator (A) domain arrangement that exposed the ADP-binding site after phosphorylation. To identify the effects of protein-lipid interactions, we have now used the TR-XSS technique to register kinetics and structural dynamics in a Ca2+-transporting P-type ATPase from Listeria monocytogenes (LMCA1) in nanodiscs at different lipid compositions. In negatively charged POPG lipids, the LMCA1 reaction kinetics and structural dynamics were reminiscent of the SERCA1a isoform, which is in agreement with the bacterial membranes being predominantly negatively charged. In uncharged POPC nanodiscs, the LMCA1 reaction was significantly different with slow buildup of the rate-limiting state. The results enable a mechanistic view of how protein-lipid interactions regulate the P-type ATPase reaction cycle. The TR-XSS technique presents a unique possibility to simultaneously determine kinetics and structural rearrangements in P-type ATPases to better understand the transport reactions and their regulation mechanisms.

Structural Basis for the Binding of Allosteric Activators Leucine and ADP to Mammalian Glutamate Dehydrogenase.

Glutamate dehydrogenase (GDH) plays a key role in the metabolism of glutamate, an important compound at a cross-road of carbon and nitrogen metabolism and a relevant neurotransmitter. Despite being one of the first discovered allosteric enzymes, GDH still poses challenges for structural characterization of its allosteric sites. Only the structures with ADP, and at low (3.5 Å) resolution, are available for mammalian GDH complexes with allosteric activators. Here, we aim at deciphering a structural basis for the GDH allosteric activation using bovine GDH as a model. For the first time, we report a mammalian GDH structure in a ternary complex with the activators leucine and ADP, co-crystallized with potassium ion, resolved to 2.45 Å. An improved 2.4-angstrom resolution of the GDH complex with ADP is also presented. The ternary complex with leucine and ADP differs from the binary complex with ADP by the conformation of GDH C-terminus, involved in the leucine binding and subunit interactions. The potassium site, identified in this work, may mediate interactions between the leucine and ADP binding sites. Our data provide novel insights into the mechanisms of GDH activation by leucine and ADP, linked to the enzyme regulation by (de)acetylation.

Prediction of protein mononucleotide binding sites using AlphaFold2 and machine learning

In this study, we developed a system that predicts the binding sites of proteins for five mononucleotides (AMP, ADP, ATP, GDP, and GTP). The system comprises two machine learning (ML)-based predictors using a convolutional neural network and a gradient boosting machine, two template-based predictors based on sequence and structure alignment, and a predictor that performs ensemble learning of these four predictors. In this study, data augmentation of ligand binding sites with similar ligand structures was performed. For example, in the prediction of ADP-binding sites using ML methods, the binding sites of AMP and ATP, which have similar structures, are considered. In addition, we constructed the structure models using AlphaFold2, a highly accurate protein prediction method. The secondary structure and dihedral angle information obtained using the model structures were used as ML predictor features. Additionally, in the template-based predictor, the structures of the binding sites were used as templates to be explored based on structure alignment to identify the binding site of the target. Consequently, the template-based predictor based on structure alignment showed the best performance among the four individual predictors, and the ensemble predictor achieved the best performance, with an area under the curve of 0.958 for all mononucleotides.

Understanding the P-Loop Conformation in the Determination of Inhibitor Selectivity Toward the Hepatocellular Carcinoma-Associated Dark Kinase STK17B.

As a member of the death-associated protein kinase family of serine/threonine kinases, the STK17B has been associated with diverse diseases such as hepatocellular carcinoma. However, the conformational dynamics of the phosphate-binding loop (P-loop) in the determination of inhibitor selectivity profile to the STK17B are less understood. Here, a multi-microsecond length molecular dynamics (MD) simulation of STK17B in the three different states (ligand-free, ADP-bound, and ligand-bound states) was carried out to uncover the conformational plasticity of the P-loop. Together with the analyses of principal component analysis, cross-correlation and generalized correlation motions, secondary structural analysis, and community network analysis, the conformational dynamics of the P-loop in the different states were revealed, in which the P-loop flipped into the ADP-binding site upon the inhibitor binding and interacted with the inhibitor and the C-lobe, strengthened the communication between the N- and C-lobes. These resulting interactions contributed to inhibitor selectivity profile to the STK17B. Our results may advance our understanding of kinase inhibitor selectivity and offer possible implications for the design of highly selective inhibitors for other protein kinases.

Identification of a new splice-acceptor mutation in HFM1 and functional analysis through molecular docking in nonobstructive azoospermia.

To investigate the genetic cause of nonobstructive azoospermia (NOA). We performed whole exome sequencing (WES) on the proband who had three relatives suffering from NOA. We used a list of candidate genes which have high expression level in testis and their mutations have been reported in NOA. Sanger sequencing verified the identified variant and its structural and functional consequence was evaluated by protein three-dimensional (3D) structure prediction and protein-ligand docking. WES revealed a novel splice-acceptor mutation (c.1832-2A>T) in helicase for meiosis 1 (HFM1) gene, which co-segregated with the NOA in this family. 3D structural models were generated and verified. Molecular docking indicated that the c.1832-2A>T mutation affects not only the ADP binding residues but also the hydrogen bond interactions. The ADP binding site will be lost in the mutant protein, potentially causing defective crossover and synapsis. We report that the c.1832-2A>T mutation is the likely cause of NOA in the family studied. Regarding that many reported NOA genes are involved in the formation of crossovers and synapsis and have critical roles in the production of germ cells, we suggest that such genes should be considered for screening of infertility among large cohorts of infertile individuals.

Anthraquinone derivatives as ADP-competitive inhibitors of liver pyruvate kinase

Liver pyruvate kinase (PKL) is a major regulator of metabolic flux and ATP production during liver cell glycolysis and is considered a potential drug target for the treatment of non-alcoholic fatty liver disease (NAFLD). In this study, we report the first ADP-competitive PKL inhibitors and identify several starting points for the further optimization of these inhibitors. Modeling and structural biology guided the optimization of a PKL-specific anthraquinone-based compound. A structure–activity relationship study of 47 novel synthetic derivatives revealed PKL inhibitors with half-maximal inhibitory concentration (IC50) values in the 200 nM range. Despite the difficulty involved in studying a binding site as exposed as the ADP site, these derivatives feature expanded structural diversity and chemical spaces that may be used to improve their inhibitory activities against PKL. The obtained results expand the knowledge of the structural requirements for interactions with the ADP-binding site of PKL.

Mechanistic insights into multiple-step transport of mitochondrial ADP/ATP carrier

The ADP/ATP carrier (AAC) is crucial for mitochondrial functions by importing ADP and exporting ATP across the inner mitochondrial membrane. However, the mechanism of highly specific ADP recognition and transport by AAC remains largely elusive. In this work, spontaneous ADP binding process to the ground c-state AAC was investigated through rigorous molecular dynamics simulations of over 31 microseconds in total. With improved simulation strategy, we have successfully identified a highly specific ADP binding site in the upper region of the cavity, and this site exhibits selectivity for ADP over ATP based on free-energy calculations. Sequence analyses on adenine nucleotide transporters also suggest that this subgroup uses the upper region of the cavity, rather than the previously proposed central binding site located at the bottom of the cavity to discriminate their substrates. Identification of the new site unveils the unusually high substrate specificity of AAC and explains the dependence of transport on the flexibility between anti and syn glycosidic conformers of ADP. Moreover, this new site together with the central site supports early biochemical findings. In light of these early findings, our simulations described a multi-step model in which the carrier uses different sites for substrate attraction, recognition and conformational transition. These results provide new insights into the transport mechanism of AAC and other adenine nucleotide transporters.

Why do cells need oxygen? Insights from mitochondrial composition and function

Mitochondrial membrane-embedded redox proteins are classically perceived as deterministic "electron transport chain" (ETC) arrays cum proton pumps; and oxygen is seen as an "immobile terminal electron acceptor." This is untenable because: (1) there are little free protons to be pumped out of the matrix; (2) proton pumping would be highly endergonic; (3) ETC-chemiosmosis-rotary ATP synthesis proposal is "irreducibly complex"/"non-evolvable" and does not fit with mitochondrial architecture or structural/distribution data of the concerned proteins/components; (4) a plethora of experimental observations do not conform to the postulates/requisites; for example, there is little evidence for viable proton-pumps/pH-gradient in mitochondria, trans-membrane potential (TMP) is non-fluctuating/non-trappable, oxygen is seen to give copious "diffusible reactive (oxygen) species" (DRS/DROS) in milieu, etc. Quite contrarily, the newly proposed murburn model's tenets agree with known principles of energetics/kinetics, and builds on established structural data and reported observations. In this purview, oxygen is needed to make DRS, the principal component of mitochondrial function. Complex V and porins respectively serve as proton-inlet and turgor-based water-exodus portals, thereby achieving organellar homeostasis. Complexes I to IV possess ADP-binding sites and their redox-centers react/interact with O2 /DRS. At/around these complexes, DRS cross-react or activate/oxidize ADP/Pi via fast thermogenic one-electron reaction(s), condensing to form two-electron stabilized products (H2 O2 /H2 O/ATP). The varied architecture and distribution of components in mitochondria validate DRS as (i) the coupling agent of oxidative reactions and phosphorylations, and (ii) the primary reason for manifestation of TMP in steady-state. Explorations along the new precepts stand to provide greater insights on mitochondrial function and pathophysiology.

Validating the predictions of murburn model for oxygenic photosynthesis: Analyses of ligand-binding to protein complexes and cross-system comparisons

In this second half of our treatise on oxygenic photosynthesis, we provide support for the murburn model of the light reaction of photosynthesis and ratify key predictions made in the first part. Molecular docking and visualization of various ligands of quinones/quinols (and their derivatives) with PS II/Cytochrome b 6 f complexes did not support chartered 2e-transport role of quinols. A broad variety of herbicides did not show any affinity/binding-based rationales for inhibition of photosynthesis. We substantiate the proposal that disubstituted phenolics (perceived as protonophores/uncouplers or affinity-based inhibitors in the classical purview) serve as interfacial modulators of diffusible reactive (oxygen) species or DR(O)S. The DRS-based murburn model is evidenced by the identification of multiple ADP-binding sites on the extra-membraneous projection of protein complexes and structure/distribution of the photo/redox catalysts. With a panoramic comparison of the redox metabolic machinery across diverse organellar/cellular systems, we highlight the ubiquitous one-electron murburn facets (cofactors of porphyrin, flavin, FeS, other metal centers and photo/redox active pigments) that enable a facile harnessing of the utility of DRS. In the summative analyses, it is demonstrated that the murburn model of light reaction explains the structures of membrane supercomplexes recently observed in thylakoids and also accounts for several photodynamic experimental observations and evolutionary considerations. In toto, the work provides a new orientation and impetus to photosynthesis research. Communicated by Ramaswamy H. Sarma

Molecular movie of nucleotide binding to a motor protein

BackgroundThe SecA DEAD (Asp-Glu-Ala-Asp) motor protein uses binding and hydrolysis of adenosine triphosphate (ATP) to push secretory proteins across the plasma membrane of bacteria. The reaction coordinate of nucleotide exchange is unclear at the atomic level of detail. MethodsWe performed multiple atomistic computations of the DEAD motor domain of SecA with different occupancies of the nucleotide and magnesium ion sites, for a total of ~1.7 μs simulation time. To characterize dynamics at the active site we analyzed hydrogen-bond networks. ResultsATP and ADP can bind spontaneously at the interface between the nucleotide binding domains, albeit at an intermediate binding site distinct from the native site. Binding of the nucleotide is facilitated by the presence of a magnesium ion close to the glutamic group of the conserved DEAD motif. In the absence of the magnesium ion, protein interactions of the ADP molecule are perturbed. ConclusionsA protein hydrogen-bond network whose dynamics couples to the occupancy of the magnesium ion site helps guide the nucleotide along the nucleotide exchange path. In SecA, release of magnesium might be required to destabilize the ADP binding site prior to release of the nucleotide. General significanceWe identified dynamic hydrogen-bond networks that help control nucleotide exchange in SecA, and stabilize ADP at an intermediate site that could explain slow release. The reaction coordinate of the protein motor involves complex rearrangements of a hydrogen-bond network at the active site, with perturbation of the magnesium ion site likely occurring prior to the release of ADP.

Computational model to analyze and characterize the functional mutations of NOD2 protein causing inflammatory disorder - Blau syndrome.

Blau syndrome (BS), which affects the eyes, skin, and joints, is an autosomal dominant genetic inflammatory disorder. BS is caused by mutations in the NOD2 gene. However, there are no direct treatments, and treatment with conventional anti-inflammatory drugs such as adrenal glucocorticoids, anti-metabolites, and biological agents such as anti-TNF and infliximab have all been attempted with varying degrees of success. In this study, we tried to identify all the reported mutations in the NOD2 protein that cause BS. Collectively, 114 missense mutations were extracted from the UniProt, ClinVar, and HGMD databases. The mutations were further subjected to pathogenic, stability, and conservation analyses. According to these computational analyses, six missense mutations (R334Q, R334W, E383G, E383K, R426H, and T605P) were found to be highly deleterious, destabilizing, and positioned in the conserved position. ADP to ATP conversion plays a crucial role in switching the closed-form of NOD2 protein to the open-form, thus activating the protein. Accordingly, the mutations in the ADP binding sites have received more attention in comparison to the mutations in the non-ADP binding positions. Interestingly, the W490L mutation is positioned in the ADP binding site and exhibits highly deleterious and destabilizing properties. Additionally, W490L was also found to be conserved, with a ConSurf score of 7. Therefore, we further performed homology modeling to determine the 3D structure of native NOD2 and the W490L mutant. Molecular docking analysis was carried out to understand the change in the interaction of ADP with the NOD2 protein. We observed that ADP had a stronger interaction with the native NOD2 protein compared to the W490L mutant. Finally, ADP complexed with native NOD2 and W490L mutant were subjected to molecular dynamics simulations, and the trajectories were analyzed. In the simulations, we observed decreased deviation and fluctuations in native NOD2, whereas decreased compactness and inter- and intramolecular hydrogen bonds were observed in the W490L mutant. This study is expected to serve as a platform for developing targeted drug therapy for BS.

Discovery of benzo[cd]indol-2-one and benzylidene-thiazolidine-2,4-dione as new classes of NLRP3 inflammasome inhibitors via ER-β structure based virtual screening

The structure-guided virtual screening (VS) has proved to be successful strategy in identification of new scaffolds for biological targets. The overactivity of NLRP3 inflammasome has been implicated in variety of inflammatory diseases including Alzheimer's disease. The up-regulation of estrogen-receptor β (ER-β) activity has been directly linked with inhibition of NLRP3 inflammasome activity. In the present study, we report discovery of new NLRP3 inflammasome inhibitors via ER-β crystal structure (PDB: 5TOA) guided virtual screening of 20,000 compound library. For experimental validation, top 10 ligands were selected based on structure novelty, docking score, prime MMGB/SA binding affinity and interaction pattern analysis. Amongst the tested compounds, three thiazolidin-4-ones IIIM-1268, IIIM-1269 and IIIM-1270 and benzo[cd]indol-2-one IIIM-1266 have shown 73, 69, 75 and 77% suppression of IL-1β release in mouse macrophages (J774A.1 cells) at 10 µM. Benzylidene-thiazolidine-2,4-diones IIIM-1268 and IIIM-1270 inhibited IL-1β release with IC50 of 2.3 and 3.5 µM and also significantly decreased the protein expression level of mature form of IL-1β in western-blot analysis. IIIM-1266 and IIIM-1270 displayed bidentate H-bonding with Arg 346 and Glu 305 residues in the active site of ER-β; and they also strongly occupied the ADP-binding site of NLRP3 protein. The results presented herein, indicate that ER-β guided VS can be successfully used to identify new NLRP3 inflammasome inhibitors, which may have potential in the development of novel anti-Alzheimer agents.

Insilico Characterization, Phylogenetic Analysis and Homology based 3D Structural Modeling of HSPA4 Heat Shock Protein Family a (Hsp70) Member 4 from Homo Sapiens (Human)

HSP70 are the specialized Heat Shock Proteins that show their novelty presence inside the deepest part of Cellular Component Networks of Chaperons and involve with various heat Shock repairing up mechanisms as main Catalytic Components. They actively participate in proper alignment and resistance of all types pressures by their proper dealing with substrates and proper exposure of their hydrophobic peptide portions inside of their substrate molecules. The exchange of information cum process of information occurs between the cell components through HSP70’s low level of affinity ATP bind sites and High Affinity level of ADP binding sites. Therefore, ATP hydrolysis process plays a key role in fundamental mechanisms, in vitro and as well as in in vivo repairing mechanisms for the functioning of Chaperone like functionalities of HSP 70 proteins. In the current study analysis, Homology Modeling methods permits us for modeling the 3D structure of the protein by taking backbone of experimentally determined 3D-Structures of homologous proteins extracted in various structural formats like PDBs. We worked with HSPA4 heat shock protein family A (HSP 70) part 4 from Homo sapiens ( Human ) and conducted 3-D structure prediction using an Automated Swiss Model Generation by employing the crystal structures of Template molecule. PDB Blast and Template search for required protein were conducted with help of all the parameters with respect to Imapact and Psi Impact angles and that leads to for most noteworthy sequence identity, structural alignments and functionalities. Finally, the homology modeling were performed by using SWISS modeler and modeled proteins were fine tuned by using the Ramachandran Plot, by using PROCHECK and 3D-Check Software Programs.

Insights into the inhibitory mechanisms of NADH on the \u03b1\u03b3 heterodimer of human NAD-dependent isocitrate dehydrogenase

Human NAD-dependent isocitrate dehydrogenase (NAD-IDH) catalyzes the oxidative decarboxylation of isocitrate in the citric acid cycle. In the α2βγ heterotetramer of NAD-IDH, the γ subunit plays the regulatory role and the β subunit the structural role. Previous biochemical data have shown that mammalian NAD-IDHs can be inhibited by NADH; however, the molecular mechanism is unclear. In this work, we show that the αβ, αγ and α2βγ enzymes of human NAD-IDH can be inhibited by NADH, and further determine the crystal structure of the αγ heterodimer bound with an Mg2+ and an NADH at the active site and an NADH at the allosteric site, which resembles that of the inactive αMgγ heterodimer. The NADH at the active site occupies the binding site for NAD+ and prevents the binding of the cofactor. The NADH at the allosteric site occupies the binding sites for ADP and citrate and blocks the binding of the activators. The biochemical data confirm that the NADH binding competes with the binding of NAD+ and the binding of citrate and ADP, and the two effects together contribute to the NADH inhibition on the activity. These findings provide insights into the inhibitory mechanisms of the αγ heterodimer by NADH.

Zinc inhibition of pyruvate kinase of M-type isozyme

Inhibitory effect of Zn on the pyruvate kinase of M (muscle)-type isozyme was analyzed for the purpose of elucidating the cytotoxicity of Zn. Zn inhibited pyruvate kinase uncompetitively with respect to the substrate PEP, and competitively with respect to ADP. Quotient velocity plot calculated from the Zn-inhibition curves showed that Zn2+ as a ZnADP complex acted as competitive and uncompetitive inhibitors of the enzyme with respect to the substrate ADP and PEP, respectively: Zn2+ forms a ZnADP complex, which may bind to the ADP-binding site of the free enzyme with the Ki value of 1.4μM causing competitive inhibition, or to the ADP-site of the enzyme-PEP complex with 2.6μM resulting in uncompetitive inhibition. The inhibition of pyruvate kinase by Zn2+ may be responsible for the cytotoxicity of this metal by decreasing glycolytic flux.

Molecular cloning, purification and characterization of Brugia malayi phosphoglycerate kinase

Phosphoglycerate kinase (PGK) is a glycolytic enzyme present in many parasites. It has been reported as a candidate molecule for drug and vaccine developments. In the present study, a full-length cDNA encoding the Brugia malayi 3-phosphoglycerate kinase (BmPGK) with an open reading frame of 1.3 kb was isolated and PCR amplified and cloned. The exact size of the BmPGK's ORF is 1377 bps. The BmPGK gene was subcloned into pET-28a (+) expression vector, the expressed enzyme was purified by affinity column and characterized. The SDS–PAGE analysis revealed native molecular weight of recombinant Brugia malayi 3-phosphoglycerate kinase (rBmPGK) to be ∼45 kDa. The enzyme was found sensitive to temperature and pH, it showed maximum activity at 25 °C and pH 8.5. The Km values for PGA and ATP were 1.77 and 0.967 mM, respectively. The PGK inhibitor, clorsulon and antifilarial drugs albendazole and ivermectin inhibited the enzyme. The specific inhibitor of PGK, clorsulon, competitively inhibited enzyme with Ki value 1.88 μM. Albendazole also inhibited PGK competitively with Ki value 35.39 μM. Further these inhibitory studies were confirmed by docking and molecular simulation of drugs with enzyme. Clorsulon interacted with substrate binding site with glutamine 37 as well as in hinge regions with aspartic acid 385 and valine 387 at ADP binding site. On the other hand albendazole interacted with asparagine 335 residues. These effects were in good association with binding interactions. Thus current study might help in designing and synthesis of effective inhibitors for this novel drug target and understanding their mode of interaction with the potent anthelmintic drugs.

A fluorometric study of modification of bovine serum albumin with structural analogues of taurine chloramine

We studied the effects of taurine chloramine and its structural analogues, antiplatelet agents, on the tertiary structure of bovine serum albumin by recording changes in its fluorescence spectrum. BSA was chosen as a model of the extracellular part of the purine P2Y12 receptor in platelets. For the detection of weak spectral shifts of the protein fluorescence, the index, which represents the ratio of the two light sums measured from approximately the middle of the spectrum towards the short- and longwave limits of its registration, was used. Administration of N-chlorotaurine and its analogues N-acetyl-N-chlorotaurine and N-isopropyl-N-chlorotaurine in albumin solution in equimolar concentrations cause weakening and red shift of the tryptophanyl fluorescence of the protein, indicating reorganization of the protein’s tertiary structure, thereby changing the properties of the microenvironment of tryptophan residues, primarily, Trp 134. All these changes are probably due to a covalent modification of the sulfhydryl group of Cys34 residue. It was hypothesized that chloramines of taurine have the ability to inhibit the activity of the ADP P2Y12 receptor in platelets due to the modification of its sulfhydryl group and that the inhibitory effect on the ADP-binding site has an allosteric character.

Dissimilar properties of pyruvate kinase from rabbit and hare muscle

Some catalytic and kinetic properties of pyruvate kinase (PK, EC 2.7.1.40) isolated from rabbit and hare heart and skeletal muscle with a 9–16-fold degree of purification were studied. The initial specific activity of the enzyme in hare heart homogenates was by 66% while in hare skeletal muscle by 25% higher than in respective rabbit tissues. Temperature optimums and thermostability of PK from hare tissues were higher as compared with those in rabbits. A comparison of KM (S0.5) values shows that PK from hare skeletal muscle exhibits a highest affinity to phosphoenol pyruvate, but lowest to ADP, as compared with PK from rabbit skeletal muscle. Moreover, PK from both hare tissues exhibits a positive kinetic cooperativity (Hill coefficient > 1.35) of the phosphoenol pyruvate and ADP binding sites. In contrast to PK from rabbit tissues, the enzyme from hare heart and skeletal muscle is more sensitive to the inhibiting effect of ATP. It is suggested that in hare skeletal muscle PK is presented by its allosteric isoform which might be advantageous under extreme conditions of the hare’s habitation.