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B6db references: 99230292

type Journal Article
authors Steegborn, C.; Clausen, T.; Sondermann, P.; Jacob, U.; Worbs, M.; Marinkovic, S.; Huber, R.; Wahl, M. C.
title Kinetics and inhibition of recombinant human cystathionine gamma-lyase. Toward the rational control of transsulfuration
journal J Biol Chem
ui 99230292
year (1999)
volume 274
number 18
pages 12675-84.
keywords Amino Acid Sequence
abstract 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.
last changed 2008/04/02 19:23

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