Kinetic analysis and chemical modification studies of nicotinate phosphoribosyltransferase from yeast
Nicotinate phosphoribosyltransferase (NaPRTase) from Baker's yeast catalyzes the formation of nicotinate mononucleotide (NaMN) and pyrophosphate from phosphoribosyl {alpha}-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 ({sup 14}C)-ADP. This rate of exchange was inhibited by PRibPP and pyrophosphate. Incubations of NaPRTase with pyridoxal 5{prime}-phosphate followed by sodium borohydride reduction led to inactivation of the enzyme. Pyridoxal was a less effective inhibitor than pyridoxal 5{prime}-phosphate. The inactivation of the enzyme by pyridoxal 5{prime}-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{sup 2+}, provided protection against this inactivation, while a kinetic analysis revealed the inhibition to be competitive, and noncompetitive, respectively. One mole of ({sup 3}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{prime}-phosphate was observed in the combination of both of the two substrates.
- Research Organization:
- City Univ. of New York, NY (USA)
- OSTI ID:
- 6795197
- Country of Publication:
- United States
- Language:
- English
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Related Subjects
59 BASIC BIOLOGICAL SCIENCES
ADP
ATP
BIOCHEMICAL REACTION KINETICS
CARBON 14 COMPOUNDS
CHEMICAL COMPOSITION
ENZYME INHIBITORS
ENZYMES
EUMYCOTA
FUNGI
HYDROGEN COMPOUNDS
ISOTOPE APPLICATIONS
KINETICS
LABELLED COMPOUNDS
MICROORGANISMS
NUCLEOTIDES
ORGANIC COMPOUNDS
OXYGEN COMPOUNDS
PHOSPHATES
PHOSPHORUS COMPOUNDS
PLANTS
REACTION KINETICS
TRACER TECHNIQUES
TRANSFERASES
TRITIUM COMPOUNDS
YEASTS