THE HUMAN TRIFUNCTIONAL ENZYME OF DE-NOVO PURINE BIOSYNTHESIS - HETEROLOGOUS EXPRESSION, PURIFICATION, AND PRELIMINARY CHARACTERIZATION

The cDNA for the human trifunctional enzyme of de novo purine biosynth esis, which encodes glycinamide ribonucleotide synthetase, aminoimidaz ole ribonucleotide synthetase, and glycinamide ribonucleotide trans-fo rmylase, has been overexpressed in Escherichia coli and its protein pr oduct has been purified to homogeneity, The glycinamide ribonucleotide transformylase activity, which constitutes the C-terminal domain of t he trifunctional enzyme, has been characterized with respect to its ki netic constants, V-max = 3.03 +/- 0.15 mu mol/min-mg and K-m values fo r beta-glycinamide ribonucleotide and 10-formyl-5,8-dideazafolate of 0 .94 +/- 0.21 and 1.58 +/- 0.25 mu M, respectively, and its kinetic mec hanism, which is ordered-sequential with the folate substrate binding first, The correspondence of these data to those obtained for the glyc inamide ribonucleotide transformylase activity of the mammalian trifun ctional enzyme indicates that the recombinant enzyme is fully function al. (C) 1998 Academic Press.