Biomedical subjects

I Tsigelny

Publications and source records attributed to I Tsigelny.

31 records · Page 2Linked to original sources

PubMed1996

Aspartate 74 as a primary determinant in acetylcholinesterase governing specificity to cationic organophosphonates.

Through site-specific mutagenesis, we examined the determinants on acetylcholinesterase which govern the specificity and reactivity of three classes of substrates: enantiomeric alkyl phosphonates, trifluoromethyl acetophenones, and carboxyl esters. By employing cationic and uncharged pairs of enantiomeric alkyl methylphosphonyl thioates of known absolute stereochemistry, we find that an aspartate residue near the gorge entrance (D74) is responsible for the enhanced reactivity of the cationic organophosphonates. Removal of the charge with the mutation D74N causes a near equal reduction in the reaction rate constants for the Rp and Sp enantiomers and exerts a greater influence on the cationic organophosphonates than on the charged trimethylammonio trifluoromethyl acetophenone and acetylthiocholine. This pattern of reactivity suggests that the orientation of the leaving group for both enantiomers is directed toward the gorge exit and in apposition to Asp 74. Replacement of tryptophan 86 with alanine in the choline subsite also diminishes the reaction rates for cationic organophosphonates, although to a lesser extent than with the D74N mutation, while not affecting the reactions with the uncharged compounds. Hence, reaction with cationic OPs depends to a lesser degree on Trp 86 than on Asp 74. Docking of Sp and Rp cycloheptyl methylphosphonyl thiocholines and thioethylates in AChE as models of the reversible complex and transition state using molecular dynamics affords structural insight into the spatial arrangement of the substituents surrounding phosphorus prior to and during reaction. The leaving group of the Rp and Sp enantiomers, regardless of charge, is directed to the gorge exit and toward Asp 74, an orientation unique to tetrahedral ligands.

Acetylcholinesterase↗

PubMed1996

Catalytic subunit of cAMP-dependent protein kinase: electrostatic features and peptide recognition.

The electrostatic field was calculated for the mammalian cAMP-dependent protein kinase (PKA) catalytic subunit (C-subunit) complexed with a 20-residue peptide from a heat stable protein kinase inhibitor (PKI: 5-24). The electrostatic field was also calculated for the C-subunit complexed with a modeled heptapeptide substrate that has been used extensively in structure/function studies for the C-subunit. Perturbations in the electrostatic free energy were calculated when single ionizable active site residues were mutated to alanine. These perturbations in electrostatic free energy were correlated to changes in the binding energy measured in a charge-to-alanine scan of the homologous yeast C-subunit by M. J. Zoller and C. S. Gibbs [(1991) Journal of Biological Chemistry, Vol. 266, pp. 8923-8931; C. S. Gibbs and M. J. Zoller (1991) Biochemistry, Vol. 30, p. 22]. This analysis indicated that the substrate binding parameters primarily depend on electrostatic interactions between a substrate or inhibitor and the C-subunit. Amino acid replacements that led to large perturbations in the electrostatic field are listed in the text. pKa shifts were also calculated for the substrate's phosphate accepting atom, the serine hydroxyl oxygen, when the active site ionizable residues were changed to structurally similar uncharged amino acids. The theoretical mutation of three active site residues caused large shifts in this parameter: E91Q, D166N, and D184N. The calculated pKa shifts for these mutants indicate that the rate of phosphotransfer should be markedly reduced in these cases. This prediction has been experimentally confirmed for the D166N mutant. The correlation between calculated electrostatic free energy changes and measured binding energy, and pKa shifts with phosphotransfer for C-subunit mutants were within experimental error of the measurements. The calculations of electrostatic energy and delta pKa have identified previously unconsidered active site residues in the mammalian C-subunit that contribute to binding energy and phosphotransfer.

Animals↗

PubMed1996

Examination of an active-site electrostatic node in the cAMP-dependent protein kinase catalytic subunit.

The active site of the cAMP-dependent protein kinase catalytic subunit harbors a cluster of acidic residues-Asp 127, Glu 170, Glu 203, Glu 230, and Asp 241-that are not conserved throughout the protein kinase family. Based on crystal structures of the catalytic subunit, these amino acids are removed from the site of phosphoryl transfer and are implicated in substrate recognition. Glu 230, the most buried of these acidic residues, was mutated to Ala (rC[E230A]) and Gln (rC[E230Q]) and overexpressed in Escherichia coli. In contrast to the mostly insoluble and destabilized rC[E230A], rC[E230Q] is largely soluble, purifies like wild-type enzyme, and displays wild-type-like thermal stability. The mutation in rC[E230Q] causes an order of magnitude decrease in the affinity for a heptapeptide substrate, Kemptide. In addition, two independent kinetic techniques were used to dissect phosphoryl transfer and product release steps in the reaction pathway. Viscosometric and pre-steady-state quench-flow analyses revealed that the phosphoryl transfer rate constant decreases by an order of magnitude, whereas the product release rate constant remains unperturbed. Electrostatic alterations in the rC[E230Q] active site were assessed using modeling techniques that provide molecular interpretations for the substrate affinity and phosphoryl transfer rate decreases observed experimentally. These observations indicate that subsite recognition elements in the catalytic subunit make electrostatic contributions that are important not only for peptide affinity, but also for catalysis. Protein kinases may, therefore, discriminate substrates by not only binding them tightly, but also by only turning over ones that complement the electrostatic character of the active site.

Animals↗

PubMed1995

Structures stabilizing the dimer interface on human 11 beta-hydroxysteroid dehydrogenase types 1 and 2 and human 15-hydroxyprostaglandin dehydrogenase and their homologs.

Human 11 beta-hydroxysteroid dehydrogenase-types 1 and 2 and human 15-hydroxyprostaglandin dehydrogenase belong to a large family of oxidoreductases that includes human dihydropteridine reductase and Streptomyces hydrogenans 20 beta-hydroxysteroid dehydrogenase, for which 3D structures are available. Almost all of these enzymes are either dimers or tetramers. The dimer interface of rat dihydropteridine reductase consists of alpha-helices E and F from each monomer arranged in a four alpha-helix bundle [Varughese et al. (1992) Proc. Natl. Acad. Sci. USA 89, 6080-6084]. Alpha-helix F contains tyrosine-146 and lysine-150, residues that are highly conserved in this protein superfamily and have been proposed to be at the catalytic site. We have examined the dimer interface between alpha-helix F in human and rat dihydropteridine reductase and Streptomyces hydrogenans 20 beta-hydroxysteroid dehydrogenase as well as modeled 3D structures of steroid and prostaglandin dehydrogenases and homologs for stabilizing interactions. We find a site in the middle of alpha-helix F that stabilizes the dimer. This anchor is adjacent to conserved lysine on alpha-helix F. Our analysis suggests that sequence variation in the anchor may be important in substrate specificity.

11-beta-Hydroxysteroid Dehydrogenases↗

PubMed1995

Amino acids important in enzyme activity and dimer stability for Drosophila alcohol dehydrogenase.

We have determined the nucleotide sequences of eight ethyl methanesulphonate-induced mutants in Drosophila alcohol dehydrogenase (ADH), of which six were previously characterized by Hollocher and Place [(1988) Genetics 116, 253-263 and 265-274]. Four of these ADH mutants contain a single amino acid change: glycine-17 to arginine, glycine-93 to glutamic acid, alanine-159 to threonine, and glycine-184 to aspartic acid. Although these mutants are inactive, three mutants (Gly17Arg, Gly93Glu and Gly184Asp) form stable homodimers, as well as heterodimers with wild-type ADH, in which the wild-type ADH subunit retains full enzyme activity [Hollocher and Place (1988) Genetics 116, 265-274]. Interestingly, the Ala159Thr mutant does not form either stable homodimers or heterodimers with wild-type ADH, suggesting that alanine-159 is important in stabilizing ADH dimers. The mutations were analysed in terms of a three-dimensional model of ADH using bacterial 20 beta-hydroxysteroid dehydrogenase and rat dihydropteridine reductase as templates. The model indicates that mutations in glycine-17 and glycine-93 affect the binding of NAD+. It also shows that alanine-159 is part of a hydrophobic anchor on the dimer interface of ADH. Replacement of alanine-159 with threonine, which has a larger side chain and can hydrogen bond with water, is likely to reduce the strength of the hydrophobic interaction. The three-dimensional model shows that glycine-184 is close to the substrate binding site. Replacement of glycine-184 with aspartic acid is likely to alter the position of threonine-186, which we propose hydrogen bonds to the carboxamide moiety of NAD+. Also, the negative charge on the aspartic acid side chain may interact with the substrate and/or residues in the substrate binding site. These mutations provide information about ADH catalysis and the stability of dimers, which may also be useful in understanding homologous dehydrogenases, which include the human 17 beta-hydroxysteroid, 11 beta-hydroxysteroid and 15-hydroxyprostaglandin dehydrogenases.

Alcohol Dehydrogenase↗

PubMed1995

Amino acid residues controlling reactivation of organophosphonyl conjugates of acetylcholinesterase by mono- and bisquaternary oximes.

Single and multiple site mutants of recombinant mouse acetylcholinesterase (rMoAChE) were inhibited with racemic 7-(methylethoxyphosphinyloxy)-1-methylquinolinium iodide (MEPQ) and the resulting mixture of two enantiomers, CH3PR,S(O)(OC2H5)-AChE(EMPR,S-AChE), were subjected to reactivation with 2-(hydroxyiminomethyl)-1-methylpyridinium methanesulfonate (P2S) and 1-(2'-hydroxyiminomethyl-1'-pyridinium)-3-(4"-carbamoyl-1"- pyridinium)-2-oxapropane dichloride (HI-6). Kinetic analysis of the reactivation profiles revealed biphasic behavior with an approximate 1:1 ratio of two presumed reactivatable enantiomeric components. Equilibrium dissociation and kinetic rate constants for reactivation of site-specific mutant enzymes were compared with those obtained for wild-type rMoAChE, tissue-derived Torpedo AChE and human plasma butyrylcholinesterase. Substitution of key amino acid residues at the entrance to the active-site gorge (Trp-286, Tyr-124, Tyr-72, and Asp-74) had a greater influence on the reactivation kinetics of the bisquaternary reactivator HI-6 compared with the monoquaternary reactivator P2S. Replacement of Phe-295 by Leu enhanced reactivation by HI-6 but not by P2S. Of residues forming the choline-binding subsite, the E202Q mutation had a dominant influence where reactivation by both oximes was decreased 16- to 33-fold. Residues Trp-86 and Tyr-337 in this subsite showed little involvement. These kinetic findings, together with energy minimization of the oxime complex with the phosphonylated enzyme, provide a model for differences in the reactivation potencies of P2S and HI-6. The two kinetic components of oxime reactivation of MEPQ-inhibited AChEs arise from the chirality of O-ethyl methylphosphonyl moieties conjugated with Ser-203 and may be attributable to the relative stability of the phosphonyl oxygen of the two enantiomers in the oxyanion hole.

Acetylcholinesterase↗

PubMed1995

Theoretical analysis of the structure of the peptide fasciculin and its docking to acetylcholinesterase.

The fasciculins are a family of closely related peptides that are isolated from the venom of mambas and exert their toxic action by inhibiting acetylcholinesterase (AChE). Fasciculins belong to the structural family of three-fingered toxins from Elapidae snake venoms, which include the alpha-neurotoxins that block the nicotinic acetylcholine receptor and the cardiotoxins that interact with cell membranes. The features unique to the known primary and tertiary structures of the fasciculin molecule were analyzed. Loop I contains an arginine at position 11, which is found only in the fasciculins and could form a pivotal anchoring point to AChE. Loop II contains five cationic residues near its tip, which are partly charge-compensated by anionic side chains in loop III. By contrast, the other three-fingered toxins show full charge compensation within loop II. The interaction of fasciculin with the recognition site on acetylcholinesterase was investigated by estimating a precollision orientation followed by determination of the buried surface area of the most probable complexes formed, the electrostatic field contours, and the detailed topography of the interaction surface. This approach has led to testable models for the orientation and site of bound fasciculin.

Acetylcholinesterase↗

PubMed1995

Structures important in mammalian 11 beta- and 17 beta-hydroxysteroid dehydrogenases.

We have used the X-ray crystallographic structures of rat and human dihydropteridine reductase and Streptomyces hydrogenans 20 beta-hydroxysteroid dehydrogenase to model parts of the 3-dimensional structure of human 11 beta- and 17 beta-hydroxysteroid dehydrogenases. We use this information along with previous results from studies of Drosophila alcohol dehydrogenase mutants to analyze the structures in binding sites for NAD(H) and NADP(H) in 11 beta-hydroxysteroid dehydrogenase-types 1 and 2. We also examine the structure of an alpha-helix at catalytic site of 17 beta-hydroxysteroid dehydrogenase-types 1, 2, 3, and 4. This alpha-helix contains a highly conserved tyrosine and lysine. Adjacent to the carboxyl side of this lysine is a site proposed to be important in subunit association. We find that 11 beta- and 17 beta-hydroxysteroid dehydrogenases-type 1 have the same residues at the "anchor site" and conserve other stabilizing features, despite only 20% sequence identity between their entire sequences. Similar conservation of stabilizing structures is found in the 11 beta- and 17 beta-hydroxysteroid dehydrogenases-type 2. We suggest that interactions of the dimerization surface of alpha-helix F with proteins or membranes may be important in regulating activity of hydroxysteroid dehydrogenases.

11-beta-Hydroxysteroid Dehydrogenases↗

PubMed1994

JNK2 contains a specificity-determining region responsible for efficient c-Jun binding and phosphorylation.

The transcriptional activity of c-Jun is augmented through phosphorylation at two sites by a c-Jun amino-terminal kinase (JNK). All cells express two distinct JNK activities, 46 and 55 kD in size. It is not clear which of them is the more important c-Jun kinase and how they specifically recognize c-Jun. The 46-kD form of JNK was identified as a new member of the MAP kinase group of signal-transducing enzymes, JNK1. Here, we report the molecular cloning of the 55-kD form of JNK, JNK2, which exhibits 83% identity and similar regulation to JNK1. Despite this close similarity, the two JNKs differ greatly in their ability to interact with c-Jun. JNK2 binds c-Jun approximately 25 times more efficiently than JNK1, and as a result has a lower Km toward c-Jun than JNK1. The structural basis for this difference was investigated and traced to a small beta-strand-like region near the catalytic pocket of the enzyme. Modeling suggests that this region is solvent exposed and therefore is likely to serve as a docking site that increases the effective concentration of c-Jun near JNK2. These results explain how two closely related MAP kinases can differ in their ability to recognize specific substrates and thereby elicit different biological responses.

Amino Acid Sequence↗

PubMed1994

Adding a positive charge at residue 46 of Drosophila alcohol dehydrogenase increases cofactor specificity for NADP+.

We previously reported that the D39N mutant of Drosophila alcohol dehydrogenase (ADH), in which Asp-39 is replaced with asparagine, has a 60-fold increase in affinity for NADP+ and a 1.5-fold increase in kcat compared to wild-type ADH [Chen et al. (1991) Eur. J. Biochem. 202, 263-267] and proposed that this part of ADH is close to the 2'-phosphate on the ribose moiety of NADP+. Here we report the effect of replacing Ala-46 with an argine residue, and A46R mutant, on binding of NADP+ to ADH and its catalytic efficiency with the NADP+ cofactor, and a modeling of the three-dimensional structure of the NAD(+)-binding region of ADH. The A46R mutant has a 2.5-fold lower Km(app)NADP+ and a 3-fold higher kcat with NADP+ compared to wild-type ADH; binding of NAD+ to the mutant was unchanged and kcat with NAD+ was lowered by about 30%. For the A46R mutant, the ratio of kcat/Km of NAD+ to NADP+ is 85, over ten-fold lower than that for wild-type ADH. Our model of the 3D structure of the NAD(+)-binding region of ADH shows that Ala-46 is over 10 A from the ribose moiety of NAD+, which would suggest that there is little interaction between this residue and NAD+ and explain why its mutation to arginine has little effect on NAD+ binding. However, the positive charge at residue 46 can neutralize some of the coulombic repulsion between Asp-39 and the 2'-phosphate on the ribose moiety of NADP+, which would increase its affinity for the A46R mutant. We also constructed a double mutant, D39N/A46R mutant, which we find has a 30-fold lower Km(app)NADP+ and 8-fold higher kcat with NADP+ as a cofactor compared to wild-type ADH; binding of NAD+ to this double mutant was lowered by 5-fold and kcat was increased by 1.5-fold. As a result, kcat/Km for the double mutant was the same for NAD+ and NADP+. The principle effect of the two mutations in ADH is to alter its affinity for the nucleotide cofactor; kcat decreases slightly in A46R with NAD+ and remains unchanged or increases in the other mutants.

Alanine↗

PubMed1994

Protein kinases share a common structural motif outside the conserved catalytic domain.

A comparison of the sequences of the mammalian and Dictyostelium catalytic subunits of cAMP-dependent protein kinase revealed extensive sequence similarities through the catalytic core and the carboxy terminal tail. The amino terminal sequences however differ dramatically. The large difference in size, 73 kDa for the Dictyostelium enzyme versus 40 kDa is due to an extension in the N-terminus. The mouse enzyme has at its amino-terminus a long amphipatic helix, the A-helix, that precedes the catalytic core, covering the surface of both lobes of the enzyme. Dictyostelium does in fact, have a similar motif but it is remote from the catalytic core, in the N-terminal extension. On the basis of molecular modeling, it is proposed that residues 77-98 correspond to a structural motif similar to the A-helix in mouse catalytic subunit. Sequences encoding similar putative motifs contiguous to the catalytic core can be recognized in many other protein kinases and is particularly prominent in all of the non-receptor tyrosine kinases. In the case of Src, this A-helix motif appears to serve as the linker between the conserved catalytic core and the SH2 domain. The interaction between the A-helix motif and the core is described, and the general occurrence of this structure within the protein kinase family is discussed.

Amino Acid Sequence↗

PubMed1993

A conserved helix motif complements the protein kinase core.

Residues 40-300 of the mammalian catalytic (C) subunit of cAMP-dependent protein kinase define a conserved bilobal catalytic core shared by all eukaryotic protein kinases. Contiguous to the core is an extended amphipathic alpha-helix (A helix). Trp30, a prominent feature of this helix, fills a deep hydrophobic pocket between the two lobes on the surface opposite to the active site. The C subunit in Dictyostelium discoideum shows sequence conservation of residues 40-350 with the mouse enzyme but contains an N-terminal extension of 332 residues. A sequence corresponding to the A helix contiguous to the core is absent. However, we have now identified a remote A-helix motif (residues 77-98). When the core of the Dictyostelium C subunit was modeled, based on the mouse C subunit, complementarity between this putative A helix and the surface of the core was found to be conserved. Analysis of other protein kinases reveals that the A-helix motif is not restricted to cAMP-dependent protein kinase. In the Src-related family of protein kinases, for example, an A helix is very likely contiguous to the core, thus serving as a linker between the conserved catalytic core and the Src homology 2 domain. We predict that an A-helix motif complementary to the core will be a conserved feature of most eukaryotic protein kinases.

Amino Acid Sequence↗

PubMedDate not supplied

Delineation of selective cyclic GMP-dependent protein kinase Ialpha substrate and inhibitor peptides based on combinatorial peptide libraries on paper.

Peptide libraries on cellulose paper have proven to be valuable tools for the a priori determination of substrate specificities of cyclic AMP- and cyclic GMP-dependent protein kinases (cAMP-kinase and cGMP-kinase) on the basis of octa-peptide sequences. Here, we report the extension of our peptide library screens to 12-mer and 14-mer peptide sequences, resulting in highly cGMP-kinase Ialpha selective peptides. The sequences TQAKRKKSLAMA-amide and TQAKRKKSLAMFLR-amide, with Km values for cGMP-kinase Ialpha of 0.7 and 0.26 microM and Vmax values of 11.5 and 10.9 micromol/min/mg, respectively, display a high specificity for this enzyme. Furthermore, replacing the phosphate acceptor residue serine with alanine in TQAKRKKSLAMA-amide resulted in the highly cGMP-kinase Ialpha selective inhibitor peptide TQAKRKKALAMA-amide, with inhibitor constants for cGMP-kinase Ialpha and cAMP-kinase of 7.5 microM and 750 microM, respectively. Selective cGMP-kinase inhibitors have the potential to play an important role in the elucidation of the distinct cellular functions of cGMP-kinase separate from those activated by cAMP-kinases, and, therefore, may play an important role as pharmaceutical targets. Molecular docking experiments of the most cGMP-kinase selective sequences on a molecular model of the catalytic domain of cGMP-kinase Ialpha suggest that they adopt unique conformations, which differ significantly from those observed for the cAMP-kinase-specific inhibitor PKI(5-24). Our results suggest that despite their structural similarities, cAMP-kinase and cGMP-kinase use distinct peptide substrate and inhibitor conformations, which could account for their unique substrate specificities. These findings are further supported by cAMP- and cGMP-kinase-selective inhibitor analogs with (D)-Ala residues at the inhibitory positions.

Animals↗