Ketomethylthiobutyric acid formation from methylthioadenosine: a diffusion assay

Typical enzyme kinetics were observed when 5'-methylthioadenosine was used as substrate with extracts of malignant murine cells in a diffusion assay. The volatile product was measured after diffusion into a solution of the sulfhydryl reagent, 5,5'-dithiobis(2-nitrobenzoic acid), which it reduced to a yellow chromophore. Cysteine was required in the system. The volatile product was identified as H2S derived from the cysteine. The yield of H2S was similar to the amount of 2-keto-4-methylthiobutyric acid (KMTB) formed from methylthioadenosine when the KMTB was measured simultaneously in an ether extraction assay. KMTB could replace methylthioadenosine as a substrate capable of causing the formation of the diffusible product from cysteine. It is concluded that the following sequence of reactions takes place in the diffusion assay system: (1) 5'-methylthioadenosine + Pi leads to adenine + 5-methylthioribose-1-P, (2) 5-methylthioribose-1-P leads to KMTB, (3) KMTB + cysteine leads to methionine + 3-mercaptopyruvate, (4) 3-mercaptopyruvate + excess R-SH leads to pyruvate + H2S, (5) H2S + 5,5'-dithiobis(2-nitrobenzoic acid) leads to 5-mercapto-2-nitrobenzoic acid. Thus, the diffusion assay measures the amount of KMTB formed. The key enzyme, cysteine aminotransferase, EC 2.6.1.3, was partially purified from malignant cells and from liver and several of its characteristics are described. The diffusion assay using this enzyme is useful in measuring de novo synthesis of alpha-keto acids and it is applicable to crude enzyme preparations. The sensitivity is about 5 nmol of keto acid and the accurate range is 5 to 100 nmol.