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J. Biol. Chem., Vol. 276, Issue 21, 17851-17856, May 25, 2001

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Class II Recombinant Phosphoribosyl Diphosphate Synthase from Spinach
PHOSPHATE INDEPENDENCE AND DIPHOSPHORYL DONOR SPECIFICITY^*

Britta N. Krath and Bjarne Hove-Jensen

From the Department of Biological Chemistry, Institute of Molecular Biology, University of Copenhagen, 83H Sølvgade, DK-1307 Copenhagen K, Denmark

A recombinant form of spinach (Spinacia oleracea) phosphoribosyl diphosphate (PRPP) synthase isozyme 3 resembling the presumed mature enzyme has been synthesized in an Escherichia coli strain in which the endogenous PRPP synthase gene was deleted, and has been purified to near homogeneity. Contrary to other PRPP synthases the activity of spinach PRPP synthase isozyme 3 is independent of P_i, and the enzyme is inhibited by ribonucleoside diphosphates in a purely competitive manner, which indicates a lack of allosteric inhibition by these compounds. In addition spinach PRPP synthase isozyme 3 shows an unusual low specificity toward diphosphoryl donors by accepting dATP, GTP, CTP, and UTP in addition to ATP. The kinetic mechanism of the enzyme is an ordered steady state Bi Bi mechanism with K_ATP and K_Rib-5-P values of 170 and 110 µM, respectively, and a V_max value of 13.1 µmol (min × mg of protein)¹. The enzyme has an absolute requirement for magnesium ions, and maximal activity is obtained at 40 °C at pH 7.6.

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