Purification and characterization of rat kidney L-alanine: 4,5-dioxovalerate transaminase and inhibition by hemin

Abstract

Rat kidney l-alanine:4,5-dioxovalerate transaminase (EC 2.6.1.43), which may be involved in the formation of aminolevulinic acid in mammalian cells, was purified 82-fold to apparent homogeneity with a 19% yield. Molecular weight of the enzyme, as estimated by gel filtration, was found to be 225 000. In polyacrylamide gel electrophoresis under denaturing conditions, the enzyme moved as a single band corresponding to an M_r of 37 000, suggesting that the enzyme is composed of six identical subunits. The K_m values of l-alanine and 4,5-dioxovalerate are 2.9 and 0.25 mM, respectively. The enzyme had an optimum activity at p^H 6.6 and was most active at 65°C. Among some amino acids tested, l-alanine proved to be the most efficient amino donor, and the enzyme was also stereospecific for the l-isomer. The effect of intermediate metabolites of heme biosynthesis, for example, δ-aminolevulinic acid, protoporphyrin, hemin and bilirubin has been studied on purified l-alanine: 4,5-dioxovalerate transaminase. Amongst these metabolites, hemin and protoporphyrin were found to be effective inhibitors.