Azaserine-reactive Sulfhydryl Group of 2-Formamido-N-ribosylacetamide 5'-Phosphate:l-Glutamine Amido-ligase (Adenosine Diphosphate)

Abstract

Preliminary experiments on 2-formamido-N-ribosylacetamide 5'-phosphate : L-glutamine amido-ligase (adenosine diphosphate) (EC 6.3.5.3) (formyl-CAR' amidotransferase) that was obtained from chicken liver showed that L-asaserine-Cl4 remained bound to the enzyme after incubation of the latter with the inhibitor, treatment with sodium borohydride, and dialysis.The major radioactive product of total acid hydrolysis of the labeled protein was tentatively identified as S-carboxymethylcysteine (1).It is the purpose of this paper to present some of the evidence for attachment of azaserine to a specific cysteine residue of formyl-GAR amidotransferase that was isolated from Salmonella typhimurium (2).The major radioactive product of the degradation of labeled enzyme with papain was a derivative of azaserine linked to a dipeptide fragment of formyl-GAR amidotransferase through a thioether linkage.The final proof of the structures of some cysteine-containing degradation products of labeled enzyme, and a suggestion for the mechanism of reaction of azaserine with formyl-GAR amidotransferase, will be presented in the following paper (3). EXPERIMENTAL PROCEDURE ML-tierialsAzaserine-CleThe method of synthesis and the specific activity of this compound were described previously; 1 mpmole of labeled substance corresponds to 11,000 c.p.m.(2).Volatile Bu$ers-Triethylamine was purified according to the method of Sjiiquist (4), and was neutralized directly with acetic acid.The syrupy solution was adjusted to pH 7.2 and 4 M in triethylammonium acetate and stored at 3". Buffers of ionic strength 0.10 and pH values between 3.5 and 6.0 were made from this stock solution by further neutralization with acetic or