The bifunctional enzyme GAR-synthetase-AIR-synthetase (E2-E5) of the yeast Saccharomyces cerevisiae has been studied. The yeast strain with overproduction of E2-E5 has been obtained. The enzyme from this strain, E2-E5, has been purified and characterized. The protein is a dimer composed of two subunits with M(r) of 87 kDa. The pH and temperature optima, pH stability and thermostability for E2 and E5 have been determined. The kinetic constants for E2 and E5 have been estimated. E2 and E5 are active only in the presence of Mg2+. E5 is a K(+)-dependent enzyme as is E5 from other sources. AMP is a competitive (to ATP) inhibitor for E5; hence, in yeast cells the purine nucleotide biosynthesis de novo is regulated at the first and fifth steps. Partial chymotryptic digestion of the purified protein gives rise to two fragments with M(r) of about 40 and 46 kDa; and E2 activity remains, while that of E5 disappears in the process.