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

Komissarov, Andrey A.; Romanova, D.V.; Dmitrieva, N A; Linkova, Elena; Mironov, A. S.; Дебабов, В. Г.

doi:10.1016/0167-4838(94)90091-4

Cited by 5 publications

(4 citation statements)

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“…enter SN2 reactions [3]. Phosphate binding at the UPase active site results in the structural rearrangements that are manifestated in absorbance spectra [4]. In UPase, selectively modified with FITC, the fluorescein residue is located at the putative uridine-binding subsite and does not affect the ability to bind the second substrate, phosphate [4].…”

Section: Resultsmentioning

confidence: 99%

“…FTC-UPase was obtained as described earlier [4]. FTC-UPase (16 /IM) was incubated at 20°C with 0.16 mM SA-423 in 50 mM Tris-HCl buffer, pH 7.1.…”

Section: Methodsmentioning

confidence: 99%

“…We have investigated the effects of ionic strength variation and of a bipolar aprotic solvent on the rate of enzyme-catalyzed uridine phosphorolysis. In an attempt to elucidate the mechanism of phosphate activation at the UPase active site, we have studied the spectral properties of enzyme-bound fluorescein (which occupies an uridine-binding subsite of the UPase [4]) and have selectively modified both uridine-and phosphate-binding subsites to demonstrate that active site hydrophobicity increases on enzyme-substrate complex formation.…”

Section: Introductionmentioning

confidence: 99%

“…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.…”

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confidence: 99%

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Enzyme‐catalyzed uridine phosphorolysis: S_N2 mechanism with phosphate activation by desolvation

Komissarov¹,

Moltchan²,

Romanova³

et al. 1994

FEBS Letters

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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.

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Section: Resultsmentioning

confidence: 99%

“…FTC-UPase was obtained as described earlier [4]. FTC-UPase (16 /IM) was incubated at 20°C with 0.16 mM SA-423 in 50 mM Tris-HCl buffer, pH 7.1.…”

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

mentioning

confidence: 99%

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Enzyme‐catalyzed uridine phosphorolysis: S_N2 mechanism with phosphate activation by desolvation

Komissarov¹,

Moltchan²,

Romanova³

et al. 1994

FEBS Letters

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

et al. 1995

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Received 18 april 1995 homology to the sequence of E. coli PNP -the sequence simiAbstract Uridine phosphorylase from E. coil (Upase) has been larity being 49% and the sequence identity 25% [9]. However crystallized using vapor diffusion technique in a new monoclinic the sequence homology between Upase and other phosphorycrystal form. The structure was determined by the molecular lases is very poor. The comparison of the three-dimensional replacement method at 2.5 ~. resolution. The coordinates of the trigonal crystal form were used as a starting model and the structures of Upase and other phosphorylases has shown that refinement by the program XPLOR led to the R-factor of 18.6%.the structure of Upase and human PNP are rather similar but The amino acid fold of the protein was found to be the same as there is no any similarity between Upase and thymidine that in the trigonal crystals. The positions of flexible regions were phosphorylase structures. The study of E. coli PNP is in prorefined. The conclusion about the involvement in the active site gress and obviously its structure must be similar to that of is in good agreement with the results of the biochemical experiUpase. The Upase three-dimensional structure has been solved ments, at 3.0 ~ resolution using the data from trigonal crystals described previously [9]. This structure contains some disordered Key words: Uridine phosphorylase; E. coli; X-ray structure regions and little is known about groups involved in catalysis and/or binding at the Upase active site [2,10 12]. IntroductionIn this paper we report results of an X-ray investigation of a monoclinic form at 2.5 ~ resolution and the interpretation of the data of the selective chemical modification of the essential Uridine phosphorylase (EC 2.4.2.3; Upase) from E. coli catamino acid residues. alyzes the reversible phosphorolysis of uridine with the formation of riboso-l-phosphate and uracil [1,2]. The protein is involved in degradation of pyrimidine nucleosides and their util-2. Materials and methods ization as carbon and energy sources in E. coli cells. Upase 2.1. Crystallization along with purine nucleoside phosphorylase (PNP) and thymid-The uridine phosphorylase was crystallized in different crystal forms ine phosphorylase, belongs to the class of nucleoside by the vapor diffusion technique at room temperature [13,14]. The phosphorylases. Uridine phosphorylase has been identified as monoclinic form was obtained in sitting drops of 0.05 M Tris-HC1 the enzyme which is responsible for the cleavage of some pyrimbuffer at pH 7.3 containing 10-12 mg/ml of the protein and 4-6% PEG (mol.wt. 4,000). The equilibrium solution consisted of 0.1 M Tris-mal/ idine nucleoside analogs possessing antitumor activity. ConverNaOH pH 5.91 5.96, 20-25% PEG (mol.wt. 4,000) and 0.04% sodium sion of 5-fluorouridine (FUR) and 5-fluorodeoxyuridine azide. The crystals reached the size of 0.7 × 0.3 x 0.5 mm after 4-6 (FUdR) to 5-fluorouracil (5FU) by Upase required the usage weeks. These crystals belong to monoclinic space group P2~...

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

Komissarov

Дебабов

1995

Biochimica et Biophysica Acta (BBA) - Protein Structure and Mol

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

Cited by 5 publications

References 12 publications

Enzyme‐catalyzed uridine phosphorolysis: S_N2 mechanism with phosphate activation by desolvation

Enzyme‐catalyzed uridine phosphorolysis: S_N2 mechanism with phosphate activation by desolvation

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

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

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

Cited by 5 publications

References 12 publications

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

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

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

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

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Enzyme‐catalyzed uridine phosphorolysis: S_N2 mechanism with phosphate activation by desolvation

Enzyme‐catalyzed uridine phosphorolysis: S_N2 mechanism with phosphate activation by desolvation