Non-steady-state kinetics of brain glutamate decarboxylase resulting from interconversion of the apo- and holoenzyme.

In addition to its primary reaction, brain glutamate decarboxylase (L-glutamate 1-carboxy-lyase, EC 4.1.1.15) catalyses an alternative transamination reaction that leads to the production of apoenzyme. Apoenzyme can be converted to holoenzyme by reaction with pyridoxal 5'-phosphate, thereby completing a cyclic interconversion of the apo- and holoenzyme. The effect of the cycle on the kinetic behavior of the enzyme was investigated with the aid of a kinetic model that combines a steady-state description of the primary reaction and a non-steady-state description of the cycle. In the presence of saturating levels of the cofactor, pyridoxal 5'-phosphate, the cycle had little effect on the kinetics of slowly transaminated substrates such as glutamate. However, the kinetic behavior of aspartate, a rapidly transaminated substrate, was strongly affected by the cycle. With aspartate, a large proportion of apoenzyme was produced, resulting in non-linear decarboxylation time courses. Estimates of the steady-state kinetic parameters for aspartate (Km, Ki, Vmax) and the apparent type of inhibition were found to depend strongly on the assay time and procedure. Similar dependencies were found for the aspartate analogues, methyl alpha-DL-aspartate, cysteine sulfinate and beta-alanine, suggesting that they also undergo rapid transamination. The kinetic model accurately predicted holoenzyme levels and accurately described the decarboxylation time courses for glutamate, aspartate and mixtures of these substrates.