A continuous spectrophotometric assay for 5-aminolevulinate synthase that utilizes substrate cycling.

A continuous spectrophotometric assay for determining 5-aminolevulinic acid synthase activity is described. The assay is based upon coupling the production of coenzyme A by 5-aminolevulinic acid synthase to the reduction of NAD+ by alpha-ketoglutarate dehydrogenase and monitoring the increase in absorbance at 340 nm. Reduction of NAD+ is stoichoimetric with formation of 5-aminolevulinic acid. Kinetic parameters for glycine and succinyl-CoA are similar to those reported for other assays which measure the formation of 5-aminolevulinic acid. Regeneration of succinyl-CoA in the alpha-ketoglutarate dehydrogenase reaction facilitates determination of initial rates at subsaturating concentrations of this substrate. This assay will permit the rapid accumulation of kinetic data and aid in mechanistic analyses of both 5-aminolevulinic acid synthase and its recombinant mutants.