Class II recombinant phosphoribosyl diphosphate synthase from spinach. Phosphate independence and diphosphoryl donor specificity

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...

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Bibliographic Details
Published in:The Journal of biological chemistry 2001-05, Vol.276 (21), p.17851-17856
Main Authors: Krath, B N, Hove-Jensen, B
Format: Article
Language:English
Subjects:
Catalysis
Escherichia coli
Isoenzymes - genetics
Isoenzymes - metabolism
Kinetics
phosphoribosyl diphosphate synthase
Recombinant Proteins - genetics
Recombinant Proteins - metabolism
Ribose-Phosphate Pyrophosphokinase - genetics
Ribose-Phosphate Pyrophosphokinase - metabolism
Spinacia oleracea
Substrate Specificity
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Summary: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 micrometer, respectively, and a V(max) value of 13.1 micromol (min x mg of protein)(-1). The enzyme has an absolute requirement for magnesium ions, and maximal activity is obtained at 40 degrees C at pH 7.6.
ISSN:0021-9258
DOI:10.1074/jbc.M010172200