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

Britta N. Krath, Bjarne Hove-Jensen

    Publikation: Bidrag til tidsskriftTidsskriftsartikelForskningpeer review

    Abstract

    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 Pi, 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 KATP and KRib-5-P values of 170 and 110 μM, respectively, and a Vmax value of 13.1 μmol (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.
    OriginalsprogEngelsk
    TidsskriftJournal of Biological Chemistry
    Vol/bind276
    Udgave nummer21
    Sider (fra-til)17851-17856
    ISSN0021-9258
    DOI
    StatusUdgivet - 2001

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