OROTATE PHOSPHORIBOSYL TRANSFERASE FROM BAKER'S YEAST: I. KINETIC ANALYSIS, CHEMICAL MODIFICATION, AND PROTON NMR SPECTROSCOPY OF THE ENZYME-SUBSTRATE COMPLEX. II. AMINO ACID ANALYSIS AND NMR SPECTROSCOPY OF THE PROTEIN.
Item
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Title
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OROTATE PHOSPHORIBOSYL TRANSFERASE FROM BAKER'S YEAST: I. KINETIC ANALYSIS, CHEMICAL MODIFICATION, AND PROTON NMR SPECTROSCOPY OF THE ENZYME-SUBSTRATE COMPLEX. II. AMINO ACID ANALYSIS AND NMR SPECTROSCOPY OF THE PROTEIN.
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Identifier
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AAI8629747
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identifier
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8629747
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Creator
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STRAUSS, ROSALYN SILBERMINTZ.
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Contributor
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Donald Sloan
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Date
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1986
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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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Kinetic analysis of the effect of pH on the reversible reaction catalyzed by orotate phosphoribosyltransferase (OPRTase) from Baker's yeast revealed that different amino acid residues may enable the enzyme-catalyzed reactions to proceed in the forward and reverse directions, respectively. For the forward reaction, there appear to be at least two critical amino acid residues (pK's 4.6 and 7.1) which must be in a deprotonated state to reach a maximum activity near pH 8 which is maintained through pH 9.5. For the reverse reaction, maximum activity is reached near pH 7 (pK at 5.4) and then the activity decreases at higher pH (pK's at 7.9 and possibly above 9).;The chemical modifier diethyl pyrocarbonate (DEPC) inactivated OPRTase in a time, concentration, and pH dependent manner and substrates can partially protect OPRTase from this inactivation. Similar results were observed when para-bromophenacylbromide (pBPB) was utilized. The effect of hydroxylamine on OPRTase is also discussed.;Amino acid analysis of OPRTase revealed a protein with a composition similar to that of other PRTases but with higher percentages of histidine, serine and glycine and lower percentages of proline and valine.;A theoretical proton NMR spectrum was generated for OPRTase, based on its amino acid composition. The spectrum thus produced has a similar number of major peaks to that of the actual spectrum taken at 300 M Hz. Spectra collected at various pH values between 8 and 5, were consistent with the maintenance of the gross conformational structure of the enzyme over that pH range.;Magnetic relaxation techniques were utilized to determine the distance between the metal ion and the protons of PRPP in the Mn(II)PRPP complex, in the presence of a minimal concentration of Mn(II). Distances consistent with a more closed structure were found for Mn(II)PRPP off the enzyme whereas a more open conformation was revealed once Mn(II)PRPP was placed in the presence of 2mM OPRTase subunits.;Working models, incorporating the findings of the research described above, are proposed for the forward and reverse reactions catalyzed by OPRTase.
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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
