The Use of Human Induced Pluripotent Stem Cells for Testing Neuroprotective Activity of Pharmacological Compounds.

Development of therapeutic preparations involves several steps, starting with the synthesis of chemical compounds and testing them in different models for selecting the most effective and safest ones to clinical trials and introduction into medical practice. Cultured animal cells (both primary and transformed) are commonly used as models for compound screening. However, cell models display a number of disadvantages, including insufficient standardization (primary cells) and disruption of cell genotypes (transformed cells). Generation of human induced pluripotent stem cells (IPSCs) offers new possibilities for the development of high-throughput test systems for screening potential therapeutic preparations with different activity spectra. Due to the capacity to differentiate into all cell types of an adult organism, IPSCs are a unique model that allows examining the activity and potential toxicity of tested compounds during the entire differentiation process in vitro. In this work, we demonstrated the efficiency of IPSCs and their neuronal derivatives for selecting substances with the neuroprotective activity using two classes of compounds - melanocortin family peptides and endocannabinoids. None of the tested compounds displayed cyto- or embryotoxicity. Both melanocortin peptides and endocannabinoids exerted neuroprotective effect in the neuronal precursors and IPSC-derived neurons subjected to hydrogen peroxide. The endocannabinoid N-docosahexaenoyl dopamine exhibited the highest neuroprotective effect (~70%) in the differentiated cultures enriched with dopaminergic neurons; the effect of melanocortin Semax was ~40%. The possibility of using other IPSC derivatives for selecting compounds with the neuroprotective activity is discussed.

The fibrinolytic system: A new target for treatment of depression with psychedelics.

Current understanding of the neurobiology of depression has grown over the past few years beyond the traditional monoamine theory of depression to include chronic stress, inflammation and disrupted synaptic plasticity. Tissue plasminogen activator (tPA) is a key factor that not only promotes fibrinolysis via the activation of plasminogen, but also contributes to regulation of synaptic plasticity and neurogenesis through plasmin-mediated activation of a probrain derived neurotrophic factor (BDNF) to mature BDNF. ProBDNF activation could potentially be supressed by competition with fibrin for plasmin and tPA. High affinity binding of plasmin and tPA to fibrin could result in a decrease of proBDNF activation during brain inflammation leading to fibrosis further perpetuating depressed mood. There is a paucity of data explaining the possible role of the fibrinolytic system or aberrant extravascular fibrin deposition in depression. We propose that within the brain, an imbalance between tPA and urokinase plasminogen activator (uPA) and plasminogen activator inhibitor-1 (PAI-1) and neuroserpin favors the inhibitors, resulting in changes in neurogenesis, synaptic plasticity, and neuroinflammation that result in depressive behavior. Our hypothesis is that peripheral inflammation mediates neuroinflammation, and that cytokines such as tumor necrosis factor alpha (TNF-α) can inhibit the fibrinolytic system by up- regulating PAI-1 and potentially neuroserpin. We propose that the decrement of the activity of tPA and uPA occurs with downregulation of uPA in part involving the binding and clearance from the surface of neural cells of uPA/PAI-1 complexes by the urokinase receptor uPAR. We infer that current antidepressants and ketamine mitigate depressive symptoms by restoring the balance of the fibrinolytic system with increased activity of tPA and uPA with down-regulated intracerebral expression of their inhibitors. We lastly hypothesize that psychedelic 5-ht2a receptor agonists, such as psilocybin, can improve mood through anti- inflammatory and pro-fibrinolytic effects that include blockade of TNF-α activity leading to decreased PAI-1 activity and increased clearance. The process involves disinhibition of tPA and uPA with subsequent increased cleavage of proBDNF which promotes neurogenesis, decreased neuroinflammation, decreased fibrin deposition, normalized glial-neuronal cross-talk, and optimally functioning neuro-circuits involved in mood. We propose that psilocybin can alleviate deleterious changes in the brain caused by chronic stress leading to restoration of homeostatic brain fibrinolytic capacity leading to euthymia.

[COMPARATIVE EVALUATION OF THE TRANSGENE EXPRESSION EFFICIENCY PROVIDED BY THE MODEL GENETIC CONSTRUCTS OF DIFFERENT STRUCTURE.]

Comparative evaluation of the transgene expression efficiency provided by the model genetic constructs of different structure is an important stage in the development of new expression methods and optimization of the existing expression vectors. However, presently there is no versatile approach to this problem. The goal of this work was to suggest an experimental system for comparative evaluation of the expression efficiency provided by nonviral genetic vectors of various size and topology in human cell cultures. Such system is based on the gene of the green fluorescence protein used as a reporter as well as flow cytofluorometry for evaluation of the expression level and quantitative PCR for adequate selection of the transfection conditions. This system was tested in two model constructs: linear molecule of DNA and plasmid.

Plasminogen activator inhibitor-1 inhibits factor VIIa bound to tissue factor.

BACKGROUND AND OBJECTIVE: A growing body of experimental evidence supports broad inhibitory and regulatory activity of plasminogen activator inhibitor 1 (PAI-1). The present study was designed to investigate whether PAI-1 inhibits factor (F) VIIa complexed with tissue factor (TF), a well-known procoagulant risk factor. METHODS AND RESULTS: The ability of PAI-1 to inhibit FVIIa-TF activity was evaluated in both clotting and factor X (FX) activation assays. PAI-1 and its complex with vitronectin inhibit: (i) clotting activity of FVIIa-TF (PAI-1(IC50) , 817 and 125 nm, respectively); (ii) FVIIa-TF-mediated FX activation (PAI-1(IC50) , 260 and 50 nm, respectively); and (iii) FVIIa bound to TF expressed on the surface of stimulated endothelial cells (PAI-1(IC50) , 260 and 120 nm, respectively). The association rate constant (k(a)) for PAI-1 inhibition of FVIIa-TF was determined using a chromogenic assay. K(a) for PAI-1 inhibition of FVIIa bound to relipidated TF is 3.3-fold higher than that for FVIIa bound to soluble TF (k(a) = 0.09 ± 0.01 and 0.027 ± 0.03 µm(-1) min(-1), respectively). Vitronectin increases k(a) for both soluble and relipidated TF by 3.5- and 30-fold, respectively (to 0.094 ± 0.020 and 2.7 ± 0.2 µm(-1) min(-1)). However, only a 3.5- to 5.0-fold increase in the acylated FVIIa was observed on SDS PAGE in the presence of vitronectin for both relipidated and soluble TF, indicating fast formation of PAI-1/vitronectin/FVIIa/relipidated TF non-covalent complex. CONCLUSIONS: Our results demonstrate potential anticoagulant activity of PAI-1 in the presence of vitronectin, which could contribute to regulation of hemostasis under pathological conditions such as severe sepsis, acute lung injury and pleural injury, where PAI-1 and TF are overexpressed.

Crystal structure of an antigen-binding fragment bound to single-stranded DNA.

Antibodies to DNA are characteristic of the autoimmune disease systemic lupus erythematosus (SLE) and they also serve as models for the study of protein-DNA recognition. Anti-DNA antibodies often play an important role in disease pathogenesis by mediating kidney damage via antibody-DNA immune complex formation. The structural underpinnings of anti-DNA antibody pathogenicity and antibody-DNA recognition, however, are not well understood, due in part to the lack of direct, experimental three-dimensional structural information on antibody-DNA complexes. To address these issues for anti-single-stranded DNA antibodies, we have determined the 2.1 A crystal structure of a recombinant Fab (DNA-1) in complex with dT5. DNA-1 was previously isolated from a bacteriophage Fab display library from the immunoglobulin repertoire of an SLE-prone mouse. The structure shows that DNA-1 binds oligo(dT) primarily by sandwiching thymine bases between Tyr side-chains, which allows the bases to make sequence-specific hydrogen bonds. The critical stacking Tyr residues are L32, L49, H100, and H100A, while His L91 and Asn L50 contribute hydrogen bonds. Comparison of the DNA-1 structure to other anti-nucleic acid Fab structures reveals a common ssDNA recognition module consisting of Tyr L32, a hydrogen bonding residue at position L91, and an aromatic side-chain from the tip of complementarity determining region H3. The structure also provides a framework for interpreting previously determined thermodynamics data, and this analysis suggests that hydrophobic desolvation might underlie the observed negative enthalpy of binding. Finally, Arg side-chains from complementarity determining region H3 appear to play a novel role in DNA-1. Rather than forming ion pairs with dT5, Arg contributes to oligo(dT) recognition by helping to maintain the structural integrity of the combining site. This result is significant because antibody pathogenicity is thought to be correlated to the Arg content of anti-DNA antibody hypervariable loops.

The distal hinge of the reactive site loop and its proximity: a target to modulate plasminogen activator inhibitor-1 activity.

The serpin plasminogen activator inhibitor type 1 (PAI-1) plays a regulatory role in various physiological processes (e.g. fibrinolysis and pericellular proteolysis) and forms a potential target for therapeutic interventions. In this study we identified the epitopes of three PAI-1 inhibitory monoclonal antibodies (MA-44E4, MA-42A2F6, and MA-56A7C10). Differential cross-reactivities of these monoclonals with PAI-1 from different species and sequence alignments between these PAI-1s, combined with the three-dimensional structure, revealed several charged residues as possible candidates to contribute to the respective epitopes. The production, characterization, and subsequent evaluation of a variety of alanine mutants using surface plasmon resonance revealed that the residues His(185), Arg(186), and Arg(187) formed the major sites of interaction for MA-44E4. In contrast, the epitopes of MA-42A2F6 and MA-56A7C10 were found to be conformational. The epitope of MA-42A2F6 comprises residues Lys(243) and Glu(350), whereas the epitope of MA-56A7C10 comprises residues Glu(242), Lys(243), Glu(244), Glu(350), Asp(355), and Arg(356). The participation of Glu(350), Asp(355), and Arg(356) provides a molecular explanation for the differential exposure of this epitope in the different conformations of PAI-1 and for the effect of these antibodies on the kinetics of the formation of the initial PAI-1-proteinase complexes. The localization of the epitopes of MA-44E4, MA42A2F6, and MA-56A7C10 elucidates two previously unidentified molecular mechanisms to modulate PAI-1 activity and opens new perspectives for the rational development of PAI-1 neutralizing compounds.

Crystallization and molecular-replacement studies of a recombinant antigen-binding fragment complexed with single-stranded DNA.

Anti-DNA antibodies have been implicated in autoimmune diseases and also serve as models for understanding protein-DNA recognition. Crystals of a recombinant antigen-binding fragment (Fab) complexed with dT(5) have been obtained and initial phases have been determined using molecular replacement. The crystals diffract to 2.1 A resolution and occupy space group P6(5)22, with unit-cell parameters a = 171.8, c = 144.6 A; there are two Fabs per asymmetric unit. X-PLORdirect rotation-function calculations followed by Patterson correlation filtering were successful when using a Fab search model; however, they failed when using the individual variable and conserved domains of the Fab as search models. AMoRe successfully identified the correct solution in cases where X-PLOR failed.

Thermodynamics of Fab-ssDNA interactions: contribution of heavy chain complementarity determining region 3.

The recombinant anti-ssDNA Fab, DNA-1, and 16 heavy chain complementarity determining region 3 (HCDR3) mutant variants were selected for thermodynamic characterization of ssDNA binding. The affinity of Fab to (dT)(15) under different temperatures and cation concentrations was measured by equilibrium fluorescence quenching titration. Changes in the standard Gibbs free binding energy (DeltaG degrees ), enthalpy (DeltaH degrees ), entropy (DeltaS degrees ), and the number of ionic pairs (Z) formed upon interaction were determined. All Fab possessed an enthalpic nature of interaction with ssDNA, that was opposite to the previously reported entropically driven binding to dsDNA [Tanha, J., and Lee, J. S. (1997) Nucleic Acids Res. 25, 1442-1449]. The contribution of separate residues of HCDR3 to ssDNA interaction was investigated. Analysis of the changes in DeltaH degrees and TDeltaS degrees, induced by substitutions in HCDR3, revealed a complete entropy/enthalpy compensation. Mutations R98A and D108A at the ends of the HCDR3 loop produced increases in TDeltaS degrees ( )()by 10.4 and 15.9 kcal/mol, respectively. Substitution of proline for arginine at the top of HCDR3 resulted in a new electrostatic contact with (dT)(15). The observed linear correlation of Z and DeltaG degrees ( )()of nonelectrostatic interactions (DeltaG degrees (nonel)) at the anti-ssDNA combining site was used for the estimation of the specific DeltaG degrees (nonel) [-20 to -25 cal/(mol.A(2))], the average contact area (450-550 A(2)), the maximal Z (6-7), and the limit in affinity under standard cation concentrations [(0.5-1) x 10(8) M(-)(1)] for this family of Fab. Results suggested that rational engineering of HCDR3 could be utilized to control the affinity and likely the specificity of Ab-DNA interactions.

[A new inhibitor of pyrimidine phosphorylase]. / Novyi ingibitor pirimidinfosforilaz.

5,5-Bis(hydroxymethyl)-2-oxo-[1-(2-trifluoromethyl)-3,3,3- trifluoropropionamido)-1-trifluoromethyl-2,2,2-trifluoroethyl- 1,3,2-dioxaphosphan (CA-423) is an in vitro inhibitor of the Escherichia coli uridine and thymidine phosphorylases. Unlike widely studied nucleoside analogues, this compound binds to the enzymes irreversibly. Its LD50 in mice was 40 mg/kg. Due to the involvement of pyrimidine phosphorylases in carcinogenesis and the relatively low toxicity of CA-423, it is promising for anticancer therapy.

Site-specific mutagenesis of a recombinant anti-single-stranded DNA Fab. Role of heavy chain complementarity-determining region 3 residues in antigen interaction.

The heavy chain complementarity-determining region 3 (HCDR3) of the anti-oligo(dT) recombinant antibody fragment, DNA-1, contributes significantly to antigen binding (Komissarov, A. A., Calcutt, M. J., Marchbank, M. T., Peletskaya, E. N., and Deutscher, S. L. (1996) J. Biol. Chem. 271, 12241-12246). In the present study, the role of separate HCDR3 residues of DNA-1 in interaction with oligo(dT) was elucidated. Based on a molecular model of the combining site, residues at the base (Arg98 and Asp108) and in the middle (Tyr101-Arg-Pro-Tyr-Tyr105) of HCDR3 were predicted to support the loop conformation and directly contact the ligand, respectively. Twenty-five site-specific mutants were produced as hexahistidine-tagged proteins, purified, and examined for binding to (dT)15 using two independent methods. All mutations in the middle of HCDR3 led to either abolished or diminished affinity. Tyr101 likely participates in hydrogen bonding, while Tyr104 and Tyr105 may be involved in aromatic-aromatic interactions with the ligand. The residues Arg102 and Pro103 were not as critical as the tyrosines. It is speculated that HCDR3 interacts with the thymines, rather than the phosphates, of the ligand. A 3-fold increase in affinity was observed by mutation of Asp108 to alanine. The highly conserved Arg98 and Asp108 do not appear to form a salt bridge.

The use of Ni-nitrilotriacetic acid agarose for estimation of affinities of hexahistidine-tagged Fab to single-stranded DNA.

The complex formed between 32P-labeled (dT)15 and a hexahistidine (6-His)-tagged anti-single-stranded DNA (ssDNA) Fab, DNA-1, was trapped by addition of nickel-chelating nitrilotriacetic acid (Ni-NTA) agarose that led to efficient separation of bound ligand from free. High stability of the immobilized complex (half-life of 4 h) and low nonspecific binding of (32P](dT)15 allowed for a rapid estimation of the dissociation constant (Kd) and was found to be approximately 130 nM. Oligonucleotide bound DNA-1 preimmobilized on Ni-NTA agarose with the same Kd as the Fab/(dT)15 complex formed in solution, indicating that the interaction of the 6-His tag with the resin did not interfere with binding. Addition of unlabeled (dT)15 led to a fast exchange with bound [32P](dT)15. Mutant versions of DNA-1 were also examined and results obtained were in agreement with data from equilibrium gel filtration and fluorescence titration [A. A. Komissarov, M. J. Calcutt, M. T. Marchbank, E. N. Peletskaya, and S. L. Deutscher (1996) J. Biol. Chem. 271, 12241-12246]. These results demonstrate that the Ni-NTA assay is an efficient and accurate method to examine 6-His-tagged protein-nucleic acid complexes. Furthermore, a competition modification of this assay may be used for detection of anti-ssDNA antibodies in serum.

Stability studies of nucleic acid-binding Fab isolated from combinatorial bacteriophage display libraries.

This study examined the global stability and activity properties of recombinant DNA-binding antibody fragments that were obtained from a bacteriophage combinatorial display library. The goal of this study was to determine whether the combinatorial approach of heavy and light chain assembly in E. coli and subsequent affinity selection preferentially selects for antibody fragments with unusual structural stabilities. Specifically, the binding properties and stability of recombinant antibody fragments with or without a C-terminal His tag to temperature, pH, and guanidine-HCI were examined. Both Fab exhibited almost identical Kd (120-130, 140-170, and 450-560 nM) and maximal fluorescence quenching (20-25%) values for binding to (dT)20, (dT)15, and (dT)10, respectively. Thermal denaturation data obtained by CD spectroscopy demonstrated that both Fab possessed structural properties comparable to well-folded proteins with defined tertiary structures which were stable below 70 degrees C (Tm 73 degrees C). These results were confirmed by differential scanning calorimetry. Both Fab exhibited the same rate of irreversible thermal inactivation (0.061-0.069 min-1) at 75 degrees C and could be reversibly renatured from guanidine-HCI and pH extremes. Crystallization trials with one recombinant DNA-binding Fab yielded diffraction quality crystals also suggesting a well-defined tertiary structure.

Equilibrium binding studies of recombinant anti-single-stranded DNA Fab. Role of heavy chain complementarity-determining regions.

We previously isolated nucleic acid-binding antibody fragments (Fab) from bacteriophage display libraries representing the immunoglobulin repertoire of automimune mice to expedite the analysis of antibody-DNA recognition. In the present study, the binding properties of one such anti-DNA Fab, high affinity single-stranded (ss) DNA-binding Fab (DNA-1), were defined using equilibrium gel filtration and fluorescence titration. Results demonstrated that DNA-1 had a marked preference for oligo(dT) (100 nM dissociation constant) and required oligo(dT) >5 nucleotides in length. A detailed analysis of the involvement of the individual heavy chain (H) complementarity-determining regions (CDR) ensued using previously constructed HCDR transplantation mutants between DNA-1 and low affinity ssDNA-binding Fab (D5), a Fab that binds poorly to DNA (Calcutt, M. J. Komissarov, A. A., Marchbank, M. T., and Deutscher, S. L. (1996) Gene (Amst.) 168, 9-14). Circular dichroism studies indicated that the wild type and mutant Fab studied were of similar overall secondary structure and may contain similar combining site shapes. The conversion of D5 to a high affinity oligo(dT)-binding Fab occurred only in the presence of DNA-1 HCDR3. Results with site-specific mutants in HCDR1 further suggested a role of residue 33 in interaction with nucleic acid. The results of these studies are compared with previously published data on DNA-antibody recognition.

Analysis of a nucleic-acid-binding antibody fragment: Construction and characterization of heavy-chain complementarity-determining region switch variants.

The display of antibody (AB) fragments (Fab) on the surface of filamentous bacteriophage (phage) and selection of phage that interact with a particular antigen (Ag) has enabled the isolation of Fab that bind nucleic acids. Nucleic acid (NA) binding Ab occur in vivo in connective tissue disease patients and certain inbred strains of mice and are thought to be pathogenic. Although there is ample data concerning the amino acid (aa) sequence of murine monoclonal Ab (mAb) reactive with DNA, significantly less is known about how autoAb interact with NA. The complementarity-determining regions (CDR) contained in the Fab contribute to most Ag binding, especially through heavy (H)-chain CDR 3. We have examined the role of individual H-chain CDR of a previously isolated recombinant single-stranded DNA-binding Fab (DNA-1) in nucleic acid interaction using a combination of H-chain CDR switching and solution-binding experiments. The three H-chain CDR of DNA-1 Fab were independently switched with the H-chain CDR of a Fab (D5) with very similar sequence and framework (FR) that binds DNA poorly in order to create all possible H-chain CDR combinations. The chimeric Fab genes were bacterially expressed, and their products were purified and analyzed. Results indicated that the H-chain CDR 3 of DNA-1 Fab, in the context of the remainder of the H-chain of D5 Fab, restored binding to oligo(dT)15 to 60% of DNA-1 levels, whereas H-chain CDR 1 and 3 of DNA-1 with CDR 2 of D5 Fab restored binding to 100% A combination of H-chain CDR 2 and 3 of DNA-1 Fab with H-chain CDR 1 of D5, unexpectedly resulted in the ability of the chimeric Fab to bind RNA preferentially over DNA. These studies demonstrate the importance of both H-chain CDR 1 and 3 in DNA recognition and further suggest that the specificity of the type of NA recognized by a particular Fab can be drastically altered by exchanging CDR.

[Interaction of metal ions with the carboxyl group of Asp-5 in the active site of uridine phosphorylase from Escherichia coli K-12]. / Vzaimodeistvie ionov metallov s karboksil'noi gruppoi Asp-5 v aktivnom tsentre uridinfosforilazy iz Escherichia coli K-12.

The effects of La3+ ions on enzymatic activity and difference absorption spectra of native and fluorescein isothiocyanate (FITC) modified uridine phosphorylase from E. coli K-12 have been studied. Excess La3+, unlike Ag+, only slightly decreases the enzyme activity but provokes similar changes in the absorption spectra of both native and modified proteins. The Kd value for La3+ ions (0.2 mM) coincides with that obtained earlier for Ag+. La3+ ions (0.2 mM) have no effect on the rate of the enzyme inactivation by diethylpyrocarbonate or tetranitromethane but increases the rate of its inactivation by Woodward's reagent K (WRK). Binding of La3+ (Kd = 0.2 mM) markedly decreases the thermal stability of the enzyme which increases with a further rise in the La3+ concentration. The values of Kd (0.2 mM) as well as the difference spectra and specific interactions with WRK indicate that one of the ligands interacting with metal ions is the carboxyl group of the Asp-5 residue. According to X-ray analysis data, this residue is involved in the formation of the active center of uridine phosphorylase.

Modification with tetranitromethane of an essential tyrosine residue in uridine phosphorylase from Escherichia coli.

Treatment with tetranitromethane (TNM) rapidly and irreversibly inactivates uridine phosphorylase (UPase) from E. coli under mildly alkaline conditions. Modification of one of the four tyrosine residues decreases enzyme activity to 10%, while modification of all tyrosines decreases it to 8%. The second-order rate constant for the inactivation is 1250 +/- 50 M-1 min-1 at pH 8.0. Phosphate (0.1 M) does not affect the inactivation rate, while 5 mM uridine, or uridine plus phosphate nearly completely protect the enzyme against inactivation. Free sulfhydryl groups of UPase are not oxidized by TNM. A single modified peptide was isolated from tryptic digest by reverse-phase HPLC. The mass to charge ratio and the sequence determined are consisted with modification of Tyr-169, which corresponds to tryptic peptide 169Tyr-Asp-Thr-Tyr-Ser-Gly-Arg175. Tyrosine nitration leads to a significant decrease in the pKa of the phenolic hydroxy group without significantly affecting enzyme structure. Comparison of the pH dependence of activity and inactivation by diethylpyrocarbonate for the native and modified UPase reveals interaction between the modified tyrosine residue and an essential histidine residue (Drabikowska, A.K. and Wozniak, G (1990) Biochem. J. 270, 319-323). It is suggested that Tyr-169 takes part in the stabilization of the imidazole ring of the essential histidine in UPase.

Atomic structure at 2.5 A resolution of uridine phosphorylase from E. coli as refined in the monoclinic crystal lattice.

Uridine phosphorylase from E. coli (Upase) has been crystallized using vapor diffusion technique in a new monoclinic crystal form. The structure was determined by the molecular replacement method at 2.5 A resolution. The coordinates of the trigonal crystal form were used as a starting model and the refinement by the program XPLOR led to the R-factor of 18.6%. The amino acid fold of the protein was found to be the same as that in the trigonal crystals. The positions of flexible regions were refined. The conclusion about the involvement in the active site is in good agreement with the results of the biochemical experiments.

Complete inactivation of Escherichia coli uridine phosphorylase by modification of Asp5 with Woodward's reagent K.

Woodward's reagent K (WRK) completely inactivated Escherichia coli uridine phosphorylase by reversible binding in the active site (Ki = 0.07 mM) with subsequent modification of a carboxyl (k2 = 1.2 min-1). Neither substrate alone protected uridine phosphorylase from inactivation. The presence of phosphate did not affect the Ki and k2 values. The addition of uracil or uridine led to a significant increase of both Ki (to 2.5 or 2.1 mM, respectively) and k2 (to 6.1 or 4.8 min-1, respectively) values. Thus, WRK could react in accordance with slow (high affinity) and fast (low affinity) mechanisms. Combined addition of phosphate and uracil completely protected uridine phosphorylase. Tryptic digestion yielded a single modified peptide (Ser4-Asp(WRK)-Val-Phe-His-Leu-Gly-Leu-Thr-Lys13). Treatment of the modified enzyme with hydroxylamine led to removal of the bulky WRK residue and replacement of the Asp5 carboxyl by a hydroxamic group. The enzyme thus obtained recovered about 10% of initial specific activity, whereas its substrate binding ability changed only moderately; the Km values for phosphate and uridine were changed from 5.1 and 0.19 mM (or 7.3 and 0.14 mM according to Leer et al. (Leer, J.C., Hammer-Jespersen, K., and M. Schwartz (1977) Eur. J. Biochem. 75, 217-224)) to 22.6 and 0.12 mM, respectively. The hydroxamic enzyme had higher thermostability than the native enzyme. The results obtained demonstrated the importance of the carboxyl at position 5. The loss of activity after selective group replacement is due to impaired stabilization of the transition state rather than to a decline in substrate affinity or change of the active site structure.

Enzyme-catalyzed uridine phosphorolysis: SN2 mechanism with phosphate activation by desolvation.

The rate of uridine phosphorolysis catalyzed by uridine phosphorylase from Escherichia coli decreases with increasing ionic strength. In contrast, the rate was increased about twofold after preincubation of uridine phosphorylase with 60% acetonitrile. These data correlate with known effects of polar and bipolar aprotic solvents on SN2 nucleophilic substitution reactions. The enzyme modified with fluorescein-5'-isothiocyanate (fluorescein residue occupies an uridine-binding subsite [Komissarov et al., (1994) Biochim. Biophys. Acta 1205, 54-58]) was selectively modified with irreversible inhibitor SA-423, which reacts near the phosphate-binding subsite. The double-modified uridine phosphorylase is assumed to imitate the enzyme-substrate complex. Modification with SA-423 was accompanied with dramatic changes in the absorption spectrum of active site-linked fluorescein, which were identical to those for fluorescein in a hydrophobic medium, namely 80% acetonitrile. The data obtained suggest that an increase in active site hydrophobicity leads to phosphate desolvation and facilitates the enzymatic SN2 uridine phosphorolysis reaction.

Selective modification of putative uridine-binding site of uridine phosphorylase from E. coli with fluorescein 5'-isothiocyanate.

A putative uridine-binding site of uridine phosphorylase (EC 2.4.2.3) from E. coli was modified with fluorescein 5'-isothiocyanate (FITC). Treatment with FITC irreversibly inactivates the enzyme (Ki = 1.0 mM, k2 = 0.15 min-1). Under the conditions of 90% inactivation the incorporation of the reagent reaches about 1 mol per mol of the enzyme subunit. Addition of uridine prevents the enzyme inactivation by FITC. In contrast to this, addition of a second substrate phosphate increases the rate of inactivation by 2.3-fold (k2 = 0.34 min-1), but has no effect on the affinity of the reagent to the enzyme. The modified protein retains the ability to bind phosphate but not uridine. According to differential absorption spectroscopy data, the binding of phosphate to the active site of the enzyme is accompanied by conformational changes which may accelerate the inactivation rate. The data presented suggest that in the UPase FITC occupies the putative uridine-binding site, while the phosphate-binding site still retains the ability to interact with the second substrate.