Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.1.3.5 (5'-nucleotidase)
 3,167 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

IMP-specific, High Km 5'-nucleotidase (EC 3.1.3.5) is an ubiquitous enzyme, the activity of which is highly regulated by substrate, ATP, and inorganic phosphate. The cDNA encoding this enzyme has recently been cloned and found to contain a unique stretch of nine glutamic and four aspartic acid residues at the C-terminus. To study the effects of this acidic tail, and of ATP and inorganic phosphate on enzyme function, we generated several structural modifications of the 5'-nucleotidase cDNA, expressed the corresponding proteins in Escherichia coli and compared their molecular and kinetic properties. As with the enzyme purified from human placenta, all recombinant proteins were activated by ATP and inhibited by inorganic phosphate. Although the S0.5-values were higher, the specific activities of the purified protein variants (except that truncated at the C-terminus) were similar. The molecular mass of the full-length enzyme subunit has been estimated at 57.3 kDa and the molecular mass of the native protein, as determined by gel-filtration chromatography, was estimated to be 195 kDa. Increasing the concentration of NaCl to 0.3 M promoted oligomerization of the protein and the formation of aggregates of 332 kDa. ATP induced further oligomerization to 715 kDa, while inorganic phosphate reduced the estimated molecular mass to 226 kDa. In contrast to the truncation of 30 amino acids at the N-terminus, which did not alter enzyme properties, the removal of the polyglutamic/aspartic acid tail of 13 residues at the C-terminus caused profound kinetic and structural changes, including a 29-fold decrease in specific activity and a significant increase in the sensitivity to inhibition by inorganic phosphate in the presence of AMP. Structurally, there was a dramatic loss of the ability to form oligomers at physiological salt concentration which was only partially restored by the addition of NaCl or ATP. These data suggest an important function of the polyglutamic acid tract in the process of association and dissociation of 5'-nucleotidase subunits.
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PMID:ATP and phosphate reciprocally affect subunit association of human recombinant High Km 5'-nucleotidase. Role for the C-terminal polyglutamic acid tract in subunit association and catalytic activity. 1009 73

The crystal structure of 5'-nucleotidase (5'-NT) from E. coli, also known as UDP-sugar hydrolase, has been determined at 1.7 A resolution. Two zinc ions are present in the active site, which is located in a cleft between two domains. The dimetal center and a catalytic Asp-His dyad are the main players in the catalytic mechanism. Structure-based sequence comparisons show that the structure also provides a model for animal 5'-NTs, which together with other ectonucleotidases terminate the action of nucleotides as extracellular signaling substances in the nervous system.
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PMID:X-ray structure of the Escherichia coli periplasmic 5'-nucleotidase containing a dimetal catalytic site. 1033 72

Cytosolic 5'-nucleotidase/phosphotransferase (cN-II), specific for purine monophosphates and their deoxyderivatives, acts through the formation of a phosphoenzyme intermediate. Phosphate may either be released leading to 5'-mononucleotide hydrolysis or be transferred to an appropriate nucleoside acceptor, giving rise to a mononucleotide interconversion. Chemical reagents specifically modifying aspartate and glutamate residues inhibit the enzyme, and this inhibition is partially prevented by cN-II substrates and physiological inhibitors. Peptide mapping experiments with the phosphoenzyme previously treated with tritiated borohydride allowed isolation of a radiolabeled peptide. Sequence analysis demonstrated that radioactivity was associated with a hydroxymethyl derivative that resulted from reduction of the Asp-52-phosphate intermediate. Site-directed mutagenesis experiments confirmed the essential role of Asp-52 in the catalytic machinery of the enzyme and suggested also that Asp-54 assists in the formation of the acyl phosphate species. From sequence alignments we conclude that cytosolic 5'-nucleotidase, along with other nucleotidases, belong to a large superfamily of hydrolases with different substrate specificities and functional roles.
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PMID:Bovine cytosolic 5'-nucleotidase acts through the formation of an aspartate 52-phosphoenzyme intermediate. 1143 67

Escherichia coli 5'-nucleotidase activity is stimulated 30- to 50-fold in vitro by the addition of Co(2+). Seven residues from conserved sequence motifs implicated in the catalytic and metal-ion-binding sites of E. coli 5'-nucleotidase (Asp(41), His(43), Asp(84), His(117), Glu(118), His(217) and His(252)) were selected for modification using site-directed mutagenesis of the cloned ushA gene. On the basis of comparative studies between the resultant mutant proteins and the wild-type enzyme, a model is proposed for E. coli 5'-nucleotidase in which a Co(2+) ion may displace the Zn(2+) ion at only one of two metal-ion-binding sites; the other metal-ion-binding site retains the Zn(2+) ion already present. The studies reported herein suggest that displacement occurs at the metal-ion-binding site consisting of residues Asp(84), Asn(116), His(217) and His(252), leading to the observed increase in 5'-nucleotidase activity.
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PMID:Cobalt activation of Escherichia coli 5'-nucleotidase is due to zinc ion displacement at only one of two metal-ion-binding sites. 1260 3

Compared to the group I chaperonins such as Escherichia coli GroEL, which facilitate protein folding, many aspects of the functional mechanism of archaeal group II chaperonins are still unclear. Here, we show that monomeric forms of archaeal group II chaperonin alpha and beta from Thermoplasma acidophilum may be purified stably and that these monomers display a strong AMPase activity in the presence of divalent ions, especially Co(2+) ion, in addition to ATPase and ADPase activities. Furthermore, other nucleoside phosphates (guanosine, cytidine, uridine, and inosine phosphates) in addition to adenine nucleotides were hydrolyzed. From analyses of the products of hydrolysis using HPLC, it was revealed that the monomeric chaperonin successively hydrolyzed the phosphoanhydride and phosphoester bonds of ATP in the order of gamma to alpha. This activity was strongly suppressed by point mutation of specific essential aspartic acid residues. Although these archaeal monomeric chaperonins did not alter the refolding of MDH, their novel versatile nucleotide hydrolysis activity might fulfill a new function. Western blot experiments demonstrated that the monomeric chaperonin subunits were also present in lysed cell extracts of T. acidophilum, and partially purified native monomer displayed Co(2+)-dependent AMPase activity.
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PMID:A potentially versatile nucleotide hydrolysis activity of group II chaperonin monomers from Thermoplasma acidophilum. 1972 44