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 cellular Hep G2 RNA. Fusion to a T7 promoter allowed expression in Escherichia coli, representing the first mammalian cystathionine gamma-lyase overproduced in a bacterial system. About 90% of the heterologous gene product was insoluble, and renaturation experiments from purified inclusion bodies met with 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 activity toward L-cystathionine (Km = 0.5 mM, Vmax = 2.5 units/mg) with an optimum pH of 8.2, no residual cystathionine beta-lyase behavior and only marginal reactivity toward L-cystine and L-cysteine were detected. Inhibition studies were performed with the mechanism-based inactivators propargylglycine, trifluoroalanine, and aminoethoxyvinylglycine. Propargylglycine inactivated human cystathionine gamma-lyase much more strongly than trifluoroalanine, in agreement with the enzyme's preference for C-gamma-S bonds. Aminoethoxyvinylglycine showed slow and tight binding characteristics with a Ki of 10.5 microM, comparable with its effect on cystathionine beta-lyase. The results have important implications for the design of specific inhibitors for transsulfuration components.[1]References
- Kinetics and inhibition of recombinant human cystathionine gamma-lyase. Toward the rational control of transsulfuration. Steegborn, C., Clausen, T., Sondermann, P., Jacob, U., Worbs, M., Marinkovic, S., Huber, R., Wahl, M.C. J. Biol. Chem. (1999) [Pubmed]
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