Biosynthesis of the Purines

An enzyme-substrate (glutamine) complex was isolated from an incubation of highly purified formylglycinamide ribonucleotide amidotransferase with glutamine followed by filtration on Sephadex gel. The reaction leading to the formation of this complex was reversible and did not depend on the presence of the rest of the substrates and the metal ion cofactor (formylglycinamide ribonucleotide, ATP, and magnesium ion). Azaserine inhibits completely complex formation. The binding ratio of glutamine to the enzyme in molar units was approximately 1:1. At 2° the half-life of the complex when isolated on Sephadex G-50 was about 125 min. Analysis of the radioactive material produced from the complex after denaturation of the enzyme with acid showed that during complex formation the glutamyl moiety of glutamine was bound to the enzyme, presumably as γ-glutamyl-enzyme complex, with release of the amide nitrogen of glutamine. Ammonia could replace the amide nitrogen of glutamine as the nitrogen donor but its reactivity compared to that of glutamine was relatively low. Hydroxylamine competed with ammonia in a strictly competitive manner to inhibit the formation of the product, formylglycinamidine ribonucleotide.