Kinetic characterization of recombinant human cystathionine beta-synthase purified from E. coli.
Cystathionine beta-synthase (CBS) catalyzes the pyridoxal-50-phosphate-dependent condensation of L-serine and L-homocysteine to form L-cystathionine in the first step of the transsulfuration pathway. Although effective expression systems for recombinant human CBS (hCBS) have been developed, they require multiple chromatographic steps as well as proteolytic cleavage to remove the fusion partner. Therefore, a series of five expression constructs, each incorporating a 6-His tag, were developed to enable the efficient purification of hCBS via immobilized metal ion affinity chromatography. Two of the constructs express hCBS in fusion with a protein partner, while the others bear only the affinity tag. The addition of an amino-terminal, 6-His tag, in the absence of a protein fusion partner and in the absence or presence ofa protease-cleavable linker, was found to be sufficient for the purification of soluble hCBS and resulted in enzyme with 86-91% heme saturation and with activity similar to that reported for other hCBS expression constructs. The continuous assay for L-Cth production, employing cystathionine beta-lyase and L-lactate dehydrogenase as coupling enzymes, was employed here for the first time to determine the steady-state kinetic parameters of hCBS, via global analysis, and revealed previously unreported substrate inhibition by L-Hcys (K(i)(L-HCYS) = 2.1 +/- 0.2 mM). The kinetic parameters for the hCBS-catalyzed hydrolysis of L-Cth toL-Ser and L-Hcys were also determined and the k(cat)/K(m)(L-CTH) of this reaction is only approximately 2-fold lower than the k(cat)/K(m)(L-SER) of the physiological, condensation reaction.
Belew
Muluken S
MS
Department of Biology, Institute of Biochemistry, Carleton University, 1125 Colonel By Drive, Ottawa, Ontario K1S5B6, Canada.
Quazi
Faraz I
FI
Willmore
William G
WG
Aitken
Susan M
SM
D002384:Catalysis; D002846:Chromatography, Affinity; D003541:Cystathionine beta-Synthase / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D006418:Heme / Q000737:chemistry / Q000378:metabolism; D006639:Histidine / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D011732:Pyridoxal Phosphate / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry* / Q000302:isolation & purification*
D011994:Recombinant Proteins; D006418:Heme; D006639:Histidine; D011732:Pyridoxal Phosphate; D003541:Cystathionine beta-Synthase