OROTATE PHOSPHORIBOSYL TRANSFERASE FROM BAKER'S YEAST: STUDIES OF THE INTERACTION WITH SUBSTRATES.
Item
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Title
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OROTATE PHOSPHORIBOSYL TRANSFERASE FROM BAKER'S YEAST: STUDIES OF THE INTERACTION WITH SUBSTRATES.
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Identifier
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AAI8713743
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identifier
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8713743
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Creator
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ASHTON, ROBERT WAYNE.
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Contributor
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Donald Sloan
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Date
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1987
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Language
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English
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Publisher
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City University of New York.
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Subject
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Chemistry, Biochemistry
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Abstract
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YOPRTase has been purified from Baker's yeast to a specific activity of 400. 5-Azaorotate was shown to be utilized by YOPRTase in a metal dependent reaction via a Bi Bi Ping Pong kinetic mechanism, and has a Km value of 75.5 (+OR-) 0.1 (mu)M in this reaction. The enzymes ribokinase and PRibPP synthetase were obtained from Salmonella typhimurium. They were used to synthesise both ('14)C PRibPP and ('13)C PRibPP. HPLC assays were developed to follow the enzyme purifications and the synthesis of ('14)C PRibPP. A flow dialysis binding study with ('14)C PRibPP showed that PRibPP binds to two independent and equal sites on the YOPRTase dimer with a K(,d) of 33.3 (+OR-) 4 (mu)m. The ('13)C NMR spectrum of a solution containing YOPRTase and ('13)C PRibPP had a peak at 95 ppm which is consistent with a ('13)C-phosphoribosyl-YOPRTase intermediate. Chemical modification studies with uracil 6-aldehyde have suggested that it is a novel modifier of lysine residues in YOPRTase, and have provided evidence for the presence of lysine(s) at the active site of YOPRTase that could be involved in binding PRibPP-Mg('2+). Similar studies with pyridoxal 5-phosphate have provided evidence for the presence of one to two essential active site lysine residues per YOPRTase subunit. They appear to be involved in PRibPP-Mg('2+) binding. Finally, nonreacting HPLC gel filtration studies have shown that YOPRTase is a dimer with an apparent MW of 37,100 in a solution of 100 mM ionic strength, pH 6.8, and at protein concentrations of 0.1 to 5.0 (mu)g/ml. When PRibPP and MgCl(,2) were added to the buffer the apparent MW increased to 38,900, suggesting a conformational change in YOPRTase when PRibPP-Mg('2+) binds, but no change in the quarternary structure. OMP in the presence of MgCl(,2) appears to have no effect on the quarternary structure of YOPRTase.
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Type
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dissertation
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Source
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PQT Legacy CUNY.xlsx
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degree
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Ph.D.
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Program
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Biochemistry
