COMPARISON BETWEEN YEAST OROTATE - AND HYPOXANTHINE/GUANINE-PHOSPHORIBOSYL TRANSFERASE ACTIVITIES: NMR STUDIES OF THE CONFORMATIONS OF FREE AND BOUND 5'-PHOSPHORIBOSYL - ALPHA - 1 - PYROPHOSPHATE. KINETIC INHIBITION STUDIES WITH CHROMIUM(III) PYROPHOSPHATE.
Item
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Title
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COMPARISON BETWEEN YEAST OROTATE - AND HYPOXANTHINE/GUANINE-PHOSPHORIBOSYL TRANSFERASE ACTIVITIES: NMR STUDIES OF THE CONFORMATIONS OF FREE AND BOUND 5'-PHOSPHORIBOSYL - ALPHA - 1 - PYROPHOSPHATE. KINETIC INHIBITION STUDIES WITH CHROMIUM(III) PYROPHOSPHATE.
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Identifier
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AAI8614706
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identifier
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8614706
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Creator
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SYED, DANYAL BARKAT.
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Contributor
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Donald L. 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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This thesis describes the elucidation of Mn(II)-PRibPP conformations free in solution as well as bound to OPRTase and HGPRTase from yeast using ('1)H and ('31)P magnetic relaxation rate measurements. The eight PRibPP metal-to-nucleus distances determined were used to resolve the conformation of the binary Mn(II)-PRibPP, ternary OPRTase-Mn(II)-PRibPP and HGPRTase-Mn(II)-PRibPP complexes. In the case of the binary, Mn(II)-PRibPP complex, all the phosphates interact with the metal ion. The (alpha)- and (beta)-phosphates were observed to be directly co-ordinated, in a bidentate fashion, while 5'-PO(,4) might be co-ordinated via axial oxygen. In this conformation the (beta)-position of the C-1 carbon of ribose is essentially unavailable for on-line substitutions. In the case of the OPRTase-bound Mn(II)-PRibPP, the Mn(II)-to-(alpha) and to- (beta)-phosphate interactions were still defined as bidentate but could also occur via axial oxygen. The enzyme-bound ribose ring still exhibited a desired 2'-endo puckered conformation but was further away from the metal ion, as the 5'-phosphate lost its inner sphere axial co-ordination on OPRTase. This new position resulted in an increase of 0.8 (ANGSTROM) in the metal ion to ribose distance (as measured from the center of the ring). In the case of the HGPRTase-Mn(II)-PRibPP complex, the (alpha)- and (beta)-phosphates also remained bidentate. However, the 5'-phosphate metal distance more closely resembled the distance of Mn(II)-PRibPP than did this distance on OPRTase. Moreover, the calculated 5'-phosphate metal ion distance was consistent with a highly distorted direct co-ordination. The ribose ring of HGPRTase-bound PRibPP was also observed to position itself further away from the metal ion by a distance of 0.7 (ANGSTROM). The new PRibPP conformation thus created would seem to allow for on-line displacement at the HGPRTase active site.;Also described in this dissertation are the inhibition studies carried out on these enzyme activities with Cr(III)PP(,i) and Na(I)PP(,i) in the presence of 1-2mM Mg(II) ion. Based upon the elucidated conformations of PRibPP when bound to these enzymes, upon inhibition studies with Cr(III)PP(,i) and Na(I)PP(,i) in presence of Mg(II) and upon previous kinetic and metal activation studies, I propose that one difference between the HGPRTase and OPRTase catalyzed reactions is how these enzymes make use of several metal-ions at their active sites to perform aspects of the catalysis. (Abstract shortened with permission of author.).
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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
