PURIFICATION AND PROPERTIES OF INOSINE MONOPHOSPHATE - PYROPHOSPHATE PHOSPHORIBOSYLTRANSFERASE (ES2.4.2.8) FROM BREWERS YEAST

Inosine monophosphate (IMP):pyrophosphate phosphoribosyltransferase from brewers yeast has been purified 234-fold over the original high-speed supernatant by heat denaturation, ammonium sulfate fractionation, DEAE-cellulose chromatography, and hydroxylapatite chromatography. IMP synthesis parallels guanosine monophosphate (GMP) synthesis over the entire purification range. Considerable denaturation of the enzyme occurs below pH 5.2 and above pH 10. A broad pH optimum for activity with guanine between pH 7.0 and 8.0 and for hypoxanthine activity between pH 8.0 and 9.0 was observed. Both activities require the presence of a divalent metal ion. Optimal Mg2+ concentration for both activities is 1 × 10-3 M. The Km values at pH 7.4 and 25° are 7.7 × 10-6 M for guanine, 1.8 × 10-5 M for hypoxanthine, 2.4 × 10-5 M for 5-phosphorylribose 1-pyrophosphate (PRPP) in the presence of guanine, and 4.2 × 10-s M for PRPP in the presence of hypoxanthine. The apparent activation energies, determined by an Arrhenius plot, are 11,600 cal/mole for hypoxanthine as substrate and 5700 cal/ mole below 19° and 11,300 cal/mole above 19° for guanine as substrate. Evidence is presented which supports the concept of a single enzyme catalyzing both IMP and GMP synthesis. © 1968, American Chemical Society. All rights reserved.