Conditions for the assay of glutamate semialdehyde aminotransferase that overcome the problem of substrate instability

Abstract

Investigations into the kinetic properties of glutamate semialdehyde aminotransferase, a key enzyme in the metabolic pathway leading to chlorophyll, are made difficult by the instability of the enzyme's substrate glutamate 1-semialdehyde. The rate of spontaneous disappearance of this compound from solution is shown to vary with the square of its concentration and to be pH-dependent. Thus using conditions appropriate to the assay of the enzyme, half of the substrate is lost from solution in a few minutes. Second-order rate constants for the reaction are determined and conditions are selected whereby the effects of the spontaneous reaction are rendered insignificant. The steady-state kinetic properties of the enzyme determined using these conditions are reported.