Solvent-accessible cysteines in human cystathionine beta-synthase: crucial role of cysteine 431 in S-adenosyl-L-methionine binding.
Cystathionine beta-synthase (CBS) is a tetrameric heme protein that catalyzes the PLP-dependent condensation of serine and homocysteine to cystathionine. CBS occupies a crucial regulatory position between the methionine cycle and transsulfuration. Human CBS contains 11 cysteine residues that are highly conserved in mammals but completely absent in the yeast enzyme, which catalyzes an identical reaction, suggesting a possible regulatory role for some of these residues. In this report, we demonstrate that in both the presence and absence of the CBS allosteric regulator S-adenosyl-l-methionine (AdoMet), only C15 and C431 of human CBS are solvent accessible. Mutagenesis of C15 to serine did not affect catalysis or AdoMet activation but significantly reduced aggregation of the purified enzyme in vitro. Mutagenesis of C431 resulted in a constitutively activated form of CBS that could not be further activated by either AdoMet or thermal activation. We and others have previously reported a number of C-terminal CBS point mutations that result in a decreased or abolished response to AdoMet. In contrast to all of these previously investigated CBS mutants, the C431 mutant form of CBS was unable to bind AdoMet, indicating that either this residue is directly involved in AdoMet binding or its absence induces a conformational change that destroys the integrity of the binding site for this regulatory ligand.
Frank
Nina
N
Department of Pediatrics, University of Colorado School of Medicine at Fitzsimons, 12800 East 19th Avenue, Mail Stop 8313, P.O. Box 6511, Aurora, Colorado 80045-0511, USA.
Kery
Vladimir
V
Maclean
Kenneth N
KN
Kraus
Jan P
JP
D001665:Binding Sites; D003541:Cystathionine beta-Synthase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D003545:Cysteine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004789:Enzyme Activation; D006801:Humans; D016297:Mutagenesis, Site-Directed; D012436:S-Adenosylmethionine / Q000378:metabolism*; D020539:Sequence Analysis, Protein; D012997:Solvents / Q000737:chemistry
D012997:Solvents; D012436:S-Adenosylmethionine; D003541:Cystathionine beta-Synthase; D003545:Cysteine