Reactivity of the phosphopyridoxal groups of cystathionase

Abstract

Aminooxyacetate and alpha-amino-gamma-aminooxybutyrate (canaline) react specifically with the P-pyridoxal groups of cystathionase to produce characteristic changes in the absorption and fluorescence properties of the bound cofactor. The increase in fluorescence at 450 nm was used to monitor the reaction. Aminooxyacetate attacks the Schiff base linkage of the enzyme several times faster (k1 = 3700 M-1 min-1 and k2 = 1000 M-1 min-1) than it attacks the aldehydic carbon of free P-pyridoxal (k = 290 M-1 min-1). Similar results were obtained with canaline. The kinetic studies indicate that a Schiff base linkage in the enzyme cystathionase should offer direct kinetic advantage during the reaction between the substrate and the cofactor. It is also shown that the inhibitor L-alpha-gamma-aminobutyrate reacts with bound P-pyridoxal to form free P-pyridoxamine. The rate of formation of P-pyridoxamine parallels the rate of enzyme inactivation.