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.
Steegborn
C
C
Max-Planck-Institut für Biochemie, Abteilung Strukturforschung, Am Klopferspitz 18a, 82152 Planegg-Martinsried, Germany.
Clausen
T
T
Sondermann
P
P
Jacob
U
U
Worbs
M
M
Marinkovic
S
S
Huber
R
R
Wahl
M C
MC
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D003542:Cystathionine gamma-Lyase / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism*; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D051381:Rats; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013455:Sulfur / Q000378:metabolism*
D017931:DNA Primers; D011994:Recombinant Proteins; D013455:Sulfur; D003542:Cystathionine gamma-Lyase