Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P00790 (PGA)
 2,475 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

High-resolution crystallographic data show that Glu 168 and Glu 211 lie on opposite surfaces of the active site from Lys 345. Two different proposals for general base catalysis have emerged from these structural studies. In one scheme, the carboxylate side chains of Glu 168 and Glu 211 are proposed to ionize a trapped water molecule and the OH- serves as the base [Lebioda, L., & Stec, B. (1991) Biochemistry 30, 2817-2822]. In the other proposal, the epsilon-amino group of Lys 345 functions in general base catalysis [Wedekind, J. E., Poyner, R. R., Reed, G. H., & Rayment, I. (1994) Biochemistry 33, 9333-9342]. Genes encoding site specific mutations of these active site residues of yeast enolase, K345A, E168Q, and E211Q, have been prepared. The respective protein products of the wild type and mutant genes were expressed in Escherichia coli and isolated in homogeneous form. All three mutant proteins possess severely depressed activities in the overall reaction- < 1 part in 10(5) of wild type activity. Properties of the three mutant proteins in partial reactions were examined to define more clearly the roles of these residues in the catalytic cycle. The K345A variant fails to catalyze the exchange of the C-2 proton of 2-phospho-D-glycerate with deuterium in D2O, whereas both the E211Q and E168Q mutant proteins are functional in this partial reaction. For E211Q and E168Q enolases, exchange is essentially complete prior to appearance of product, and this observation provides further support for an intermediate in the normal reaction. K345A enolase is inactive in the ionization of tartronate semialdehyde phosphate (TSP), whereas both E168Q and E211Q proteins alter the tautomeric state or catalyze ionization of bound TSP. Wild type enolase catalyzes hydrolysis of (Z)-3-chloro-2-phosphoenolpyruvate by addition of OH- and elimination of Cl- at C-3. This reaction mimics the addition of OH- to C-3 of phosphoenolpyruvate in the reverse reaction with the normal product. All three mutant proteins are depressed in their abilities to carry out this reaction. In single-turnover assays, the activities vary in the order K345A > E168Q >> E211Q. These results suggest that Lys 345 functions as the base in the ionization of 2-PGA and that Glu 211 participates in the second step of the reaction.
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PMID:Toward identification of acid/base catalysts in the active site of enolase: comparison of the properties of K345A, E168Q, and E211Q variants. 863 1

Oxidative events that target the sugar-phosphate backbone of DNA can lead to reactive fragments that interfere with DNA repair, transcription and translation by the formation of cross-links and adducts of proteins and nucleobases. Here we report the formation of several such lesions through the aerobic degradation of an independently generated C-3'-thymidinyl radical in 2'-deoxyoligonucleotides. Individual fragments were identified by independent synthesis and comparison of retention times in high-performance liquid chromatography (HPLC) and/or matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-ToF MS) along with gel electrophoresis. The formation of this reactive intermediate in the presence of oxygen was found to produce 3'-phosphoglycolaldehyde (3'-PGA) as well as 3'-ketoenolether (3'-KEE), 3'-phosphoglycolate (3'-PG), and 5'-aldehyde terminated oligonucleotide fragments. Additionally, a significant outcome of C-3'-thymidinyl radical formation in DNA oligomers is a strand break resulting in one 3'- and two 5'-phosphate-terminated oligomers. These results suggest the involvement of several sugar derived reactive species upon C-3'-radical initiated scission of single-stranded DNA under aerobic conditions. The electrophilic nature of several of these products as well as their formation through a single oxidative event can make the presence of a C-3'-DNA radical more detrimental to the cell than products derived from more frequently occurring DNA sugar radicals.
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PMID:Aerobic fate of the C-3'-thymidinyl radical in single-stranded DNA. 1717 76