5-Phosphoribosylpyrophosphate amidotransferase from soybean root nodules: kinetic and regulatory properties

Arch Biochem Biophys. 1984 Mar;229(2):623-31. doi: 10.1016/0003-9861(84)90195-4.

Abstract

Most of the nitrogen transported from the nodules of nitrogen-fixing soybean plants is in the form of the ureides allantoin and allantoic acid. Recent work has shown that ureides are formed in the plant fraction of the nodule from de novo purine biosynthesis and purine oxidation. 5-Phosphoribosylpyrophosphate amidotransferase (PRAT), which catalyzes the first committed step of purine biosynthesis, has been purified 1500-fold from soybean root nodules. The enzyme had an apparent Mr of 8 X 10(6), but this estimate may have been for an aggregation of several purine biosynthetic activities. PRAT showed a pH optimum of pH 8.0, and Km values were 18 and 0.4 mM for glutamine and 5-phosphoribosyl-1-pyrophosphate (PRPP), respectively. The reaction required Mg2+, and PRPPMg3- was shown to be the reactive molecular species of PRPP. Ammonia could replace glutamine as a substrate, and the Vm with ammonia was twice that obtained when glutamine was the substrate. The initial-rate kinetics showed sequential addition of substrates to the enzyme. Product inhibition data was consistent with the order of product release being phosphoribosylamine, PPi, and glutamate. The enzyme was subject to regulation by end products of the purine biosynthetic pathway. IMP and GMP inhibited competitively with PRPP and promoted cooperativity in the binding of this substrate; there was no cooperativity in the binding of IMP to the enzyme. XMP was a linear competitive inhibitor with PRPP. The results are discussed in terms of the key regulatory point occupied by PRAT in the pathway of ureide biogenesis.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Amidophosphoribosyltransferase / isolation & purification
  • Amidophosphoribosyltransferase / metabolism*
  • Ammonia / pharmacology
  • Glutamine / metabolism
  • Inosine Monophosphate / pharmacology
  • Kinetics
  • Molecular Weight
  • Pentosyltransferases / metabolism*
  • Plants / enzymology*
  • Soybeans

Substances

  • Glutamine
  • Inosine Monophosphate
  • Ammonia
  • Pentosyltransferases
  • Amidophosphoribosyltransferase