Purification and characterization of the wild type and truncated human cystathionine beta-synthase enzymes expressed in E. coli.
In this paper, we describe the expression and characterization of recombinant human cystathionine beta-synthase (CBS) in Escherichia coli. We have used a glutathione-S-transferase (GST) fusion protein vector and incorporated a cleavage site with a long hinge region which allows for the independent folding of CBS and its fusion partner. In addition, our construct has the added benefit of yielding a purified CBS which only contains one extra glycine amino acid residue at the N-terminus. In our two-step purification procedure we are able to obtain a highly pure enzyme in sufficient quantities for crystallography and other physical chemical methods. We have investigated the biochemical and catalytic properties of purified full-length human CBS and of two truncation mutants lacking the C-terminal domain or both the N-terminal heme-binding and the C-terminal regulatory regions. Specifically, we have determined the pH optima of the different CBS forms and their kinetic and spectral properties. The full-length and the C-terminally truncated enzyme had a broad pH 8.5 optimum while the pH optimum of the N- and C- terminally truncated enzyme was sharp and shifted to pH 9. Furthermore, we have shown unequivocally that CBS binds one mole of heme per subunit by determining both the heme and the iron content of the enzyme. The activity of the enzyme was unaffected by the redox status of the heme iron. Finally, we show that CBS is stimulated by S-adenosyl- l-methionine but not its analogs.
Frank
Nina
N
Department of Pediatrics, University of Colorado School of Medicine, UCHSC, RC1 North, Rm. 4128, 12800, Mail Stop 8313, P.O. Box 6511, Aurora, CO 80045-0511, USA.
Kent
Jana O
JO
Meier
Markus
M
Kraus
Jan P
JP
D003541:Cystathionine beta-Synthase / Q000737:chemistry* / Q000302:isolation & purification*; D004789:Enzyme Activation; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification
D011994:Recombinant Proteins; D003541:Cystathionine beta-Synthase