Kinetics and inhibition of recombinant human cystathionine gamma-lyase - Toward the rational control of transsulfuration

The gene encoding human cystathionine gamma-lyase was cloned from total cel lular Rep G2 RNA. Fusion to a T7 promoter allowed expression in Escherichia coli, representing the first mammalian cystathionine gamma-lyase overprodu ced in a bacterial system. About 90% of the heterologous gene product was i nsoluble, and renaturation experiments from purified inclusion bodies met w ith limited success. About 5 mg/liter culture of human cystathionine gamma- lyase could also be extracted from the soluble lysis fraction, employing a three-step native procedure. While the enzyme showed high gamma-lyase activ ity toward L-cystathionine (K-m = 0.5 mM, V-max = 2.5 units/mg) with an opt imum pH of 8.2, no residual cystathionine beta-lyase behavior and only marg inal reactivity toward L-cystine and L-cysteine were detected. Inhibition s tudies were performed with the mechanism-based inactivators propargylglycin e, trifluoroalanine, and aminoethoxyvinylglycine. Propargylglycine inactiva ted human cystathionine gamma-lyase much more strongly than trifluoroalanin e, in agreement with the enzyme's preference for C-gamma-S bonds. Aminoetho xyvinylglycine showed slow and tight binding characteristics with a K-i of 10.5 mu M, comparable with its effect on cystathionine beta-lyase, The resu lts have important implications for the design of specific inhibitors for t ranssulfuration components.