Biomedical subjects

I Tsigelny

Publications and source records attributed to I Tsigelny.

At least 19 recordsLinked to original sources

PubMed2005

Coherent expression chromosome cluster analysis reveals differential regulatory functions of amino-terminal and distal parathyroid hormone-related protein domains in prostate carcinoma.

Parathyroid hormone-related protein (PTHrP) has a number of cancer-related actions. While best known for causing hypercalcemia of malignancy, it also has effects on cancer cell growth, apoptosis, and angiogenesis. Studying the actions of PTHrP in human cancer is complicated because there are three isoforms and many derived peptides. Several peptides are biologically active at known or presumed cell surface receptors; in addition, the PTHrP-derived molecules can exert effects at the cell nucleus. To address this complexity, we studied gene expression in a DU 145 prostate cancer cell line that was stably transfected with control vector, PTHrP 1-173 and PTHrP 33-173. With this model, regulatory effects of the amino-terminal portion of PTHrP would result only from transduction with the full-length molecule, while effects pertaining to distal sequences would be evident with either construct. Analysis of the expression profiles by microarrays demonstrated nonoverlapping groups of differentially expressed genes. Amino-terminal PTHrP affected groups of genes involved in apoptosis, prostaglandin and sex steroid metabolism, cell-matrix interactions, and cell differentiation, while PTHrP 33-173 caused substantial increases in MHC class I antigen expression. This work demonstrates the distinct biological actions of the amino-terminus compared to distal mid-molecule or carboxy-terminal sequences of PTHrP in prostate carcinoma cells and provides targets for further study of the malignant process.

Journal Article↗

PubMed2001

Electrostatic environment surrounding the activation loop phosphotyrosine in the oncoprotein v-Fps.

Autophosphorylation of Tyr-1073 in the activation loop of the oncoprotein v-Fps enhances the phosphoryl transfer reaction without influencing substrate, ATP, or metal ion binding affinities [Saylor, P., et al. (1998) Biochemistry 37, 17875-17881]. A structural model of v-Fps, generated from the insulin receptor, indicates that pTyr-1073 chelates two arginines. Mutation of these residues to alanine (R1042A and R1066A) results in weakly phosphorylated enzymes, indicating that one electropositive center is insufficient for attaining maximum loop phosphorylation and concomitant high catalytic activity. While the turnover rate for R1066A is similar to that for a mutant lacking a phosphorylatable residue in the activation loop, the rate for R1042A is 50-fold slower. While solvent perturbation studies suggest that the former is due to a slow phosphoryl transfer step, the latter effect results from a slow conformational change in the mutant, potentially linked to motions in the catalytic loop. Binding of a stoichiometric quantity of Mg(2+) is essential for ATP binding and catalysis, while binding of an additional Mg(2+) ion activates further the wild-type enzyme. The affinity of the R1066A enzyme for the second Mg(2+) ion is 23-fold higher than that of the phosphorylated or unphosphorylated form of wild-type v-Fps, with substrate binding unaffected. Conversely, the affinity of R1066A for a substrate mimic lacking a phosphorylation site is 12-fold higher than that for the phosphorylated or unphosphorylated form of wild-type v-Fps, with binding of the second Mg(2+) ion unaffected. A comparison of these enzyme-independent parameters indicates that Arg-1042 and Arg-1066 induce strain in the active site in the repressed form of the enzyme. While this strain is not relieved in the phosphorylated form, the improvements in catalysis in activated v-Fps compensate for reduced metal and substrate binding affinities.

Adenosine Triphosphate↗

PubMed2001

Protein docking using continuum electrostatics and geometric fit.

The computer program DOT quickly finds low-energy docked structures for two proteins by performing a systematic search over six degrees of freedom. A novel feature of DOT is its energy function, which is the sum of both a Poisson-Boltzmann electrostatic energy and a van der Waals energy, each represented as a grid-based correlation function. DOT evaluates the energy of interaction for many orientations of the moving molecule and maintains separate lists scored by either the electrostatic energy, the van der Waals energy or the composite sum of both. The free energy is obtained by summing the Boltzmann factor over all rotations at each grid point. Three important findings are presented. First, for a wide variety of protein-protein interactions, the composite-energy function is shown to produce larger clusters of correct answers than found by scoring with either van der Waals energy (geometric fit) or electrostatic energy alone. Second, free-energy clusters are demonstrated to be indicators of binding sites. Third, the contributions of electrostatic and attractive van der Waals energies to the total energy term appropriately reflect the nature of the various types of protein-protein interactions studied.

Computer Simulation↗

PubMed2000

Catalytic assessment of the glycine-rich loop of the v-Fps oncoprotein using site-directed mutagenesis.

The three glycine residues in the glycine-rich loop of the oncoprotein, v-Fps, were mutated to determine the function of these highly conserved residues in catalysis. The kinase domains of six mutants (G928A,S, G930A,S, and G933A,S) and the wild-type enzyme were expressed and purified as fusion proteins of glutathione-S-transferase in Escherichia coli, and their catalytic properties were assessed using steady-state kinetic, inhibition, viscosity and autophosphorylation studies. Although both G928A and G930A had no detectable activity toward the substrate peptide (EAEIYEAIE), the other mutants had apparent, but varying activities. G930S lowered the rate of phosphoryl transfer by 130-fold while G928S and G933S had smaller (6-9-fold) reductions in this step. These effects on catalytic function parallel the reductions in turnover and autophosphorylation but, for G933S and G933A, net product release is still rate limiting at saturating substrate and ATP concentrations. On the basis of K(I) measurements, the effects on turnover for these mutants may be due to improved ADP affinity. While ADP affinity is reduced 2- and 3-fold for G928S and G930S, the affinity of this product is increased by 22- and 7-fold for G933S and G933A. In contrast, ATP affinity is enhanced by 5-fold for G928S and G933S and is reduced by less than 2-fold for G930S. These complex, differential effects on nucleotide binding indicate that the glycines influence the relative affinities of ADP and ATP. On the basis of the results of serine replacements, Gly-928 and Gly-930 enhance ADP affinity by 9- and 2-fold compared to ATP affinity whereas Gly-933 diminishes ADP affinity by approximately 4-fold compared to ATP affinity. These findings demonstrate that the functions of the loop lie not only in modulating the rate of the phosphoryl transfer step but also in balancing the relative affinities of ATP and ADP. These effects on nucleotide specificity may be a contributing element for the stabilization of the phosphoryl transition state and may also facilitate quick release of bound products.

Adenosine Diphosphate↗

PubMed2000

Common EF-hand motifs in cholinesterases and neuroligins suggest a role for Ca2+ binding in cell surface associations.

Comparisons of protein sequence via cyclic training of Hidden Markov Models (HMMs) in conjunction with alignments of three-dimensional structure, using the Combinatorial Extension (CE) algorithm, reveal two putative EF-hand metal binding domains in acetylcholinesterase. Based on sequence similarity, putative EF-hands are also predicted for the neuroligin family of cell surface proteins. These predictions are supported by experimental evidence. In the acetylcholinesterase crystal structure from Torpedo californica, the first putative EF-hand region binds the Zn2+ found in the heavy metal replacement structure. Further, the interaction of neuroligin 1 with its cognate receptor neurexin depends on Ca2+. Thus, members of the alpha,beta hydrolase fold family of proteins contain potential Ca2+ binding sites, which in some family members may be critical for heterologous cell associations.

Amino Acid Sequence↗

PubMed2000

Subunit interface selective toxins as probes of nicotinic acetylcholine receptor structure.

The pentametric assembly of the nicotinic acetylcholine receptor with two of the five subunit interfaces serving as a ligand binding sites offers an opportunity to distinguish features on the surfaces of the subunits, and their ligand specificity characteristics. The receptor from mammalian muscle, with its circular order of homologous subunits (alphagamma alphadelta beta), assembles in a unique arrangement. The residues governing assembly can be ascertained through mutagenesis. Selectivity of certain natural toxins is sufficient to distinguish between sites at the alphagamma and alphadelta subunit interfaces. By interchanging residues on the gamma and delta subunits through mutagenesis, and ascertaining how they interact with the alpha subunit, determinants forming the binding sites can be delineated. The alpha-conotoxins show a 10,000-fold preference for the alphadelta over alphagamma subunit interface with alphaepsilon falling in between. The waglerins show a 2,000-fold preference for alphaepsilon over the alphagamma and alphadelta interfaces. Finally, the alpha-neurotoxin from N. mossambica mossambica shows a 10,000-fold preference for the alphagamma and alphadelta interfaces over alphaepsilon. Identification of interactive residues through mutagenesis, when coupled with homology modeling of domains and site-directed residue modification, has revealed important elements of receptor structure.

Animals↗

PubMed1999

600 ps molecular dynamics reveals stable substructures and flexible hinge points in cAMP dependent protein kinase.

Molecular dynamics simulations of the catalytic subunit of cAMP dependent protein kinase (cAPK) have been performed in an aqueous environment. The relations among the protein hydrogen-bonding network, secondary structural elements, and the internal motions of rigid domains were examined. The values of fluctuations of protein dihedral angles during dynamics show quite distinct maxima in the regions of loops and minima in the regions of alpha-helices and beta-strands. Analyses of conformation snapshots throughout the run show stable subdomains and indicate that these rigid domains are constrained during the dynamics by a stable network of hydrogen bonds. The most stable subdomain during the dynamics was in the small lobe including part of the carboxy-terminal tail. The most significant flexible region was the highly conserved glycine-rich loop between beta strands 1 and 2 in the small lobe. Many of the main chain dihedral angle changes measured in a comparison of the crystallographic structures of "open" and "closed" conformations of cAPK correspond to the highly flexible residues found during dynamics.

Animals↗

PubMed1999

Analysis of cholinesterase inactivation and reactivation by systematic structural modification and enantiomeric selectivity.

We show here with a congeneric series of Rp- and Sp-alkoxymethyl phosphonothiolates of known absolute stereochemistry that chiral selectivity in their reaction with acetylcholinesterase can be described in terms of discrete orientational and steric requirements. Stereoselectivity depends on acyl pocket dimensions, which govern leaving group orientation and a productive association of the phosphonyl oxygen in the oxyanion hole. Overall geometry is consistent with a pentavalent intermediate where the attacking serine and leaving group are at apical positions. Oxime reactivation of the phosphonylated enzyme occurs through a similar associative intermediate presumably forming an oxime phosphonate. The oximes of differing structure show distinct angles of attacking the phosphate where the attack angles and access to the phosphorus are constrained in the sterically impacted gorge. Hence, efficacy of oxime reactivation is dependent on both oxime and conjugated phosphonate structures.

Animals↗

PubMed1998

Mechanism of action of chromogranin A on catecholamine release: molecular modeling of the catestatin region reveals a beta-strand/loop/beta-strand structure secured by hydrophobic interactions and predictive of activity.

A novel fragment of chromogranin A, known as 'catestatin' (bovine chromogranin A344-364), inhibits catecholamine release from chromaffin cells and noradrenergic neurons by acting as a non-competitive nicotinic cholinergic antagonist, and may therefore constitute an endogenous autocrine feedback regulator of sympathoadrenal activity. To characterize how this activity depends on the peptide's structure, we searched for common 3-dimensional motifs for this primary structure or its homologs. Catestatin's primary structure bore significant (29-35.5% identity, general alignment score 44-57) sequence homology to fragment sequences within three homologs of known 3-dimensional structures, based on solved X-ray crystals: 8FAB, IPKM, and 2IG2. Each of these sequences exists in nature as a beta-strand/loop/beta-strand structure, stabilized by hydrophobic interactions between the beta-strands. The catestatin structure was stable during molecular dynamics simulations. The catestatin loop contains three Arg residues, whose electropositive side chains form the terminus of the structure, and give rise to substantial uncompensated charge asymmetry in the molecule. A hydrophobic moment plot revealed that catestatin is the only segment of chromogranin A predicted to contain amphiphilic beta-strand. Circular dichroism in the far ultraviolet showed substantial (63%) beta-sheet structure, especially in a hydrophobic environment. Alanine-substitution mutants of catestatin established a crucial role for the three central arginine residues in the loop (Arg351, Arg353, and Arg358), though not for two arginine residues in the strand region toward the amino-terminus. [125I]Catestatin bound to Torpedo membranes at a site other than the nicotinic agonist binding site. When the catestatin structure was 'docked' with the extracellular domain of the Torpedo nicotinic cholinergic receptor, it interacted principally with the beta and delta subunits, in a relatively hydrophobic region of the cation pore extracellular orifice, and the complex of ligand and receptor largely occluded the cation pore, providing a structural basis for the non-competitive nicotinic cholinergic antagonist properties of the peptide. We conclude that a homology model of catestatin correctly predicts actual features of the peptide, both physical and biological. The model suggests particular spatial and charge features of the peptide which may serve as starting points in the development of non-peptide mimetics of this endogenous nicotinic cholinergic antagonist.

Animals↗

PubMed1998

Kinetic analyses of mutations in the glycine-rich loop of cAMP-dependent protein kinase.

The conserved glycines in the glycine-rich loop (Leu-Gly50-Thr-Gly52-Ser-Phe-Gly55-Arg-Val) of the catalytic (C) subunit of cAMP-dependent protein kinase were each mutated to Ser (G50S, G52S, and G55S). The effects of these mutations were assessed here using both steady-state and pre-steady-state kinetic methods. While G50S and G52S reduced the apparent affinity for ATP by approximately 10-fold, substitution at Gly55 had no effect on nucleotide binding. In contrast to ATP, only mutation at position 50 interfered with ADP binding. These three mutations lowered the rate of phosphoryl transfer by 7-300-fold. The combined data indicate that G50 and G52 are the most critical residues in the loop for catalysis, with replacement at position 52 being the most extreme owing to a larger decrease in the rate of phosphoryl transfer (29 vs 1.6 s-1 in contrast to 500 s-1 for wild-type C). Surprisingly, all three mutations lowered the affinity for Kemptide by approximately 10-fold, although none of the loop glycines makes direct contact with the substrate. The inability to correlate the rate constant for net product release with the dissociation constant for ADP implies that other steps may limit the decomposition of the ternary product complex. The observations that G52S (a) selectively affects ATP binding and (b) significantly lowers the rate of phosphoryl transfer without making direct contact with either the nucleotide or the peptide imply that this residue serves a structural role in the loop, most likely by positioning the backbone amide of Ser53 for contacting the gamma-phosphate of ATP. Energy-minimized models of the mutant proteins are consistent with the observed kinetic consequences of each mutation. The models predict that only mutation of Gly52 will interfere with the observed hydrogen bonding between the backbone and ATP.

Amino Acid Substitution↗

PubMed1998

Identification of pairwise interactions in the alpha-neurotoxin-nicotinic acetylcholine receptor complex through double mutant cycles.

alpha-Neurotoxins are potent inhibitors of the nicotinic acetylcholine receptor (nAChR), binding with high affinity to the two agonist sites located on the extracellular domain. Previous site-directed mutagenesis had identified three residues on the alpha-neurotoxin from Naja mossambica mossambica (Lys27, Arg33, and Lys47) and four residues on the mouse muscle nAChR alpha-subunit (Val188, Tyr190, Pro197, and Asp200) as contributing to binding. In this study, thermodynamic mutant cycle analysis was applied to these sets of residues to identify specific pairwise interactions. Amino acid variants of alpha-neurotoxin from N. mossambica mossambica at position 33 and of the nAChR at position 188 showed strong energetic couplings of 2-3 kcal/mol at both binding sites. Consistently smaller yet significant linkages of 1.6-2.1 kcal/mol were also observed between variants at position 27 on the toxin and position 188 on the receptor. Additionally, toxin residue 27 coupled to the receptor residues 190, 197, and 200 at the alphadelta binding site with observed coupling energies of 1.5-1.9 kcal/mol. No linkages were found between toxin residue Lys47 and the receptor residues studied here. These results provide direct evidence that the two conserved cationic residues Arg33 and Lys27, located on loop II of the toxin structure, are binding in close proximity to the alpha-subunit region between residues 188-200. The toxin residue Arg33 is closer to Val188, where it is likely stabilized by adjacent negative or aromatic residues on the receptor structure. Lys27 is positioned closer to Tyr190, Pro197, and Asp200, where it is likely stabilized through electrostatic interaction with Asp200 and/or cation/pi interactions with Tyr190.

Amino Acid Sequence↗

PubMed1998

Toxins selective for subunit interfaces as probes of nicotinic acetylcholine receptor structure.

The pentameric structure of the nicotinic acetylcholine receptor with two of the five subunit interfaces serving as ligand binding sites offers an opportunity to distinguish features on the surfaces of the subunits and their ligand specificity characteristics. This problem has been approached through the study of assembly of subunits and binding characteristics of selective peptide toxins. The receptor, with its circular order of homologous subunits (alpha gamma alpha delta beta), assembles in only one arrangement, and through mutagenesis, the residues governing assembly can be ascertained. Selectivity of certain toxins is sufficient to readily distinguish between sites at the alpha gamma and alpha delta interfaces. By interchanging residues on the gamma and delta subunits, and ascertaining how they interact with the alpha-subunit, determinants forming the binding sites can be delineated. The alpha-conotoxins, which contain two disulfide loops and 12-14 amino acids, show a 10,000-fold preference for the alpha delta over the alpha gamma subunit interface with alpha epsilon falling between the two. The waglerins, as 22-24 amino acid peptides with a single core disulfide loop, show a 2000-fold preference for alpha epsilon over the alpha gamma and alpha delta interfaces. Finally, the 6700 Da short alpha-neurotoxin from N. mossambica mossambica shows a 10,000-fold preference for the alpha gamma and alpha delta interfaces over alpha epsilon. Selective mutagenesis enables one to also distinguish alpha-neurotoxin binding at the alpha gamma and alpha delta subunits. This information, when coupled with homology modeling of domains and site-directed residue modification, reveals important elements of receptor structure and conformation.

Amino Acid Sequence↗

PubMed1997

Role of the glycine triad in the ATP-binding site of cAMP-dependent protein kinase.

A glycine-rich loop in the ATP-binding site is one of the most highly conserved sequence motifs in protein kinases. Each conserved glycine (Gly-50, Gly-52, and Gly-55) in the catalytic (C) subunit of cAMP-dependent protein kinase (cAPK) was replaced with Ser and/or Ala. Active mutant proteins were expressed in Escherichia coli, purified to apparent homogeneity, separated into phosphoisoforms, and characterized. Replacing Gly-55 had minimal effects on steady-state kinetic parameters, whereas replacement of either Gly-50 or Gly-52 had major effects on both Km and kcat values consistent with the prediction of the importance of the tip of the glycine-rich loop for catalysis. Substitution of Gly-50 caused a 5-8-fold reduction in Km (ATP), an 8-12-fold increase in Km (peptide), and a 3-5-fold drop in kcat. The Km (ATP) and Km (peptide) values of C(G52S) were increased 8- and 18-fold, respectively, and the kcat was decreased 6-fold. In contrast to catalytic efficiency, the ATPase rates of C(G50S) and C(G52S) were increased by more than an order of magnitude. The thermostability of each mutant was slightly increased. Unphosphorylated C(G52S) was characterized as well as several isoforms phosphorylated at a single site, Ser-338. All of these phosphorylation-defective mutants displayed a substantial decrease in both enzymatic activity and thermal stability that correlated with the missing phosphate at Thr-197. These results are correlated with the crystal structure, models of the respective mutant proteins, and conservation of the Glys within the protein kinase family.

Adenosine Triphosphatases↗

PubMed1997

Synergistic binding of nucleotides and inhibitors to cAMP-dependent protein kinase examined by acrylodan fluorescence spectroscopy.

We have engineered an acrylodan-modified derivative of the catalytic subunit of cyclic AMP-dependent protein kinase (cAPK) whose fluorescence emission signal has allowed the synergistic binding between nucleotides and physiological inhibitors of cAPK to be examined (Whitehouse, S., and Walsh, D. A. (1983) J. Biol. Chem. 258, 3682-3692). In the presence of the regulatory subunit, RI, the affinity of cAPK for adenosine, ADP, AMPPNP (adenosine 5'-(beta, gamma-imino)triphosphate), or ATP was 5-, 50-, 120-, and 15,000-fold enhanced, while in the presence of the heat-stable inhibitor protein of cAPK (PKI), there was a 3-, 20-, 33-, and 2000-fold enhancement in the binding of these nucleotides, respectively. A short inhibitor peptide, PKI-(14-22), enhanced the binding of ADP to the same degree as did full-length PKI (20-fold) but, in contrast, did not significantly enhance the binding of ATP or AMPPNP. The full binding synergism between PKI and either ATP (2000-fold) or AMPPNP (33-fold) to cAPK could, however, be mimicked by a longer peptide, PKI-(5-24), suggesting that the PKI NH2 terminus (residues 5-13) is most likely critical. Since this region is remote from the ATP gamma-phosphate, the binding synergism must arise through an extended network communication mechanism between the PKI NH2 terminus and the ATP binding site.

2-Naphthylamine↗

PubMed1997

A model of the nicotinic receptor extracellular domain based on sequence identity and residue location.

We have modeled the extracellular domains of individual subunits (amino acids 31-200) in the nicotinic acetylcholine receptor using sequence homology with copper binding proteins of known crystal structure, plastocyanin and pseudoazurin, and data from recent site-specific mutagenesis, antibody mapping, and site-directed labelling studies. These data formed an initial model that was refined using molecular dynamics and mechanics as well as electrostatic and solvation energy calculations. The sequences between residues 31 and 164 in the alpha 1-subunit and corresponding residues in homologous receptor subunits show similarity with the core sequence of the cation binding site in plastocyanin and pseudoazurin, a region in the template proteins characterized by multiple hairpin loops. In addition to defining the subunit interfaces that comprise the site for agonist and competitive antagonist binding in more detail, the findings show that negatively charged residues cluster in domains arranged to diminish electrostatic free energy of the complex. Electrostatic factors also appear to distinguish the ligand binding interfaces, alpha gamma and alpha delta, from the other three interfaces on the pentameric receptor.

Amino Acid Sequence↗

PubMed1997

Solution structure of synthetic peptide inhibitor and substrate of cAMP-dependent protein kinase. A study by 2D H NMR and molecular dynamics.

Peptides derived from the inhibitor of cAMP-dependent protein kinase. PKI, have been studied by 2D 1H NMR techniques. These include the inhibitor PKI(6-22), the substrate [Ala20-Ser21]PKI(5-24), and a phosphorylated form of the latter [Ala20-Ser21P]PKI(5-24). A homologous fold was found in the three peptides which consisted of an N-terminal segment in helical conformation to residue 13 and a C-terminal segment poorly defined conformationally. A parallel study was carried out by molecular dynamics (MD) for the inhibitor peptide PKI(5-24). The N-terminal helix, as observed in the crystal structure of the catalytic subunit-PKI(5-24) complex, was conserved in the MD simulations with the enzyme-free inhibitor. Similarly the Gly14-Gly17 turn was apparent in all MD structures, whereas the C-terminal region, residues 18-24, was directed towards the N-terminal helix in contrast to the extended conformation of this segment pointing away from the N-terminal helix in the crystal structure. This is primarily due to ionic interaction between Asp9 and Arg15. Indeed, a detailed analysis of the NOE contacts by NOESY at low temperature (2 degrees C) shows the occurrence of pH-dependent contacts with Phe10. We conclude that the binding of short inhibitors, such as PKI(5-24), to the enzyme involves a conformational rearrangement of the C-terminal region. The substrate [Ala20-Ser21]PKI(5-24) and the product [Ala20-Ser21P]PKI(5-24), give very similar structures with local rearrangements involving some of the side chains.

Amino Acid Sequence↗

PubMed1997

Determining ligand orientation and transphosphonylation mechanisms on acetylcholinesterase by Rp, Sp enantiomer selectivity and site-specific mutagenesis.

Acetylcholinesterase, an enzyme of the serine hydrolase family, catalyzes the rapid hydrolysis of certain carboxyl esters. Other acyl esters efficiently transacylate the enzyme with a subsequent, slow deacylation step. Of these, the phosphoryl and phosphonyl esters are perhaps of greatest mechanistic interest since individual enantiomers of known absolute stereochemistry can be isolated and their interactions with the dissymmetric enzyme active site examined. We describe here studies of a series of enantiomeric Rp- and Sp-alkylphosphonates interacting with mouse acetylcholinesterase. Since the acetylcholinesterase is generated by recombinant DNA methods, mutant enzymes can be made with specific replacements of individual amino acid side chains. Individual amino acid replacements in the acyl pocket, the choline subsite and at the active center gorge entry have been generated, and the reaction kinetics of the mutant enzymes analyzed. These studies have shown that substitution of aliphatic amino acids for phenylalanines 295 and 297 in the acyl pocket diminishes, and in some cases, actually inverts chiral preferences. The combined structure-activity approach, where both ligand and enzyme are modified systematically, has enabled us to show that the restricted dimensions of the acyl pocket in the active center dictate enantiomeric selectivity. Moreover, the reactions of compounds of known absolute stereochemistry show three requirements for efficient transphosphonylation: (a) apposition of the phosphate with the gamma-oxygen on Ser 203 to form a pentavalent, presumed trigonal bipyramidal intermediate, (b) polarization of the phosphonyl oxygen bond by its positioning in the oxyanion hole, and (c) positioning the leaving group towards the gorge exit.

Acetylcholinesterase↗

PubMed1996

Structures important in NAD(P)(H) specificity for mammalian retinol and 11-Cis-retinol dehydrogenases.

Mammalian retinol and 11-cis retinol dehydrogenases catalyze the formation of retinaldehyde from retinol and 11-cis retinaldehyde from 11-cis retinol, respectively. Although their amino acid sequences are 54% identical, these enzymes have different cofactor specificities: rat retinol dehydrogenase uses NADP+, while cow 11-cis retinol dehydrogenase uses NAD+. We used their close sequence similarity and the availability of 3D structures of their homologs to construct a 3D model of the two retinol dehydrogenases to investigate the determinants for cofactor specificity. The structure of rat retinol dehydrogenase shows that lysine-64 is important in stabilizing binding of 2'-phosphate on NADP+ in two ways: lysine's positively charged side chain has a coulombic attraction to the 2'-phosphate and partially compensates for the negative charge of aspartic acid-38. Cow 11-cis retinol dehydrogenase has threonine-61 at the position homologous to lysine-64. Threonine-61 does not have a stabilizing coulombic interaction with NADP+, nor can threonine-61 counteract the repulsive interaction between NADP+ and aspartic acid-37 in 11-cis retinol dehydrogenase. This suggests that aspartic acid-37 and threonine-61 are important in the specificity of 11-cis retinol dehydrogenase for NAD+.

Alcohol Oxidoreductases↗