Kinetic analysis and chemical modification studies of nicotinate phosphoribosyltransferase from yeast.
Item
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Title
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Kinetic analysis and chemical modification studies of nicotinate phosphoribosyltransferase from yeast.
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Identifier
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AAI8914757
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identifier
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8914757
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Creator
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Hess, Susan Laurel.
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Contributor
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Adviser: Donald Sloan
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Date
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1988
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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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Nicotinate phosphoribosyltransferase (NaPRTase) from Baker's yeast catalyzes the formation of nicotinate mononucleotide (NaMN) and pyrophosphate from phosphoribosyl {dollar}\alpha{dollar}-1-pyrophosphate and nicotinate, concomitant with ATP hydrolysis. Using purified NaPRTase, initial velocity measurements were performed varying one substrate concentration at different fixed levels of the second substrate and maintaining the third substrate constant. Subsequently, an exchange of label was observed between ATP and ({dollar}\sp{14}{dollar}C) -ADP. This rate of exchange was inhibited by PRibPP and pyrophosphate. These results combined with other kinetic studies previously accomplished in our laboratory (Hanna, L. S., Hess, S. L., and Sloan, D. L. (1983) J. Biol. Chem. 258, 9745-9754), suggested that the enzyme proceeds through an ordered Uni Uni Bi Ter Ping Pong kinetic mechanism.;Incubations of NaPRTase with pyridoxal 5{dollar}\sp\prime{dollar}-phosphate followed by sodium borohydride reduction led to inactivation of the enzyme. Pyridoxal was a less effective inhibitor than pyridoxal 5{dollar}\sp\prime{dollar}-phosphate. The inactivation of the enzyme by pyridoxal 5{dollar}\sp\prime{dollar}-phosphate was reversible upon flow dialysis, whereas reduction of the enzyme-pyridoxal complex with sodium borohydride rendered the inhibition irreversible. The presence of ATP or PRibPP, with or with Mg{dollar}\sp{lcub}2+{rcub}{dollar}, provided protection against this inactivation, while a kinetic analysis revealed the inhibition to be competitive, and noncompetitive, respectively. One mole of ({dollar}\sp3{dollar}H) -pyridoxal phosphate was required to completely inactivate the enzyme, which was reduced in the presence of MgATP and MgPRibPP to 0.2 and 0.6, respectively. No incorporation of pyridoxal 5{dollar}\sp\prime{dollar}-phosphate was observed in the combination of both of the two substrates. Taken together, these results suggest the presence of a lysine residue at the active site of the enzyme which can form a Schiff base intermediate with pyridoxal 5{dollar}\sp\prime{dollar}-phosphate.;pH stability studies indicated the enzyme was stable over the pH range 4.1-9.6. Plots of log Vmax and log Vmax/Km vs. pH for ATP, yielded pKa values of 5.3 and 5.6, respectively. The profile of log Vmax for PRibPP yielded a pKa value of 5.8 and a pKb value of 8.6, while a plot of log Vmax/Km yielded a pKa value of 5.5. These findings suggest the presence of a carboxyl or imidazole group of an amino acid residue, in addition to a lysine residue, at the catalytic site of NaPRTase.
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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.
