Regulation of human cystathionine beta-synthase by S-adenosyl-L-methionine: evidence for two catalytically active conformations involving an autoinhibitory domain in the C-terminal region.
Cystathionine beta-synthase (CBS), condensing homocysteine and serine, represents a key regulatory point in the biosynthesis of cysteine via the transsulfuration pathway. Inherited deficiency of CBS causes homocystinuria. CBS is activated by S-adenosyl-L-methionine (AdoMet) by inducing a conformational change involving a noncatalytic C-terminal region spanning residues 414-551. We report the purification of two patient-derived C-terminal mutant forms of CBS, S466L and I435T, that provide new insight into the mechanism of CBS regulation and indicate a regulatory function for the "CBS domain". Both of these point mutations confer catalytically active proteins. The I435T protein is AdoMet inducible but is 10-fold less responsive than wild-type (WT) CBS to physiologically relevant concentrations of this compound. The S466L form does not respond to AdoMet but is constitutively activated to a level intermediate between those of WT CBS in the presence and absence of AdoMet. Both mutant proteins are able to bind AdoMet, indicating that their impairment is related to their ability to assume the fully activated conformation that AdoMet induces in WT CBS. We found that I435T and WT CBS can be activated by partial thermal denaturation but that the AdoMet-stimulated WT, S466L, and a truncated form of CBS lacking the C-terminal region cannot be further activated by this treatment. Tryptophan and PLP fluorescence data for these different forms of CBS indicate that activation by AdoMet, limited proteolysis, and thermal denaturation share a common mechanism involving the displacement of an autoinhibitory domain located in the C-terminal region of the protein.
Janosík
M
M
Department of Pediatrics, University of Colorado School of Medicine, Denver, Colorado 80262, USA.
Kery
V
V
Gaustadnes
M
M
Maclean
K N
KN
Kraus
J P
JP
D020134:Catalytic Domain; D003001:Cloning, Molecular; D003541:Cystathionine beta-Synthase / Q000037:antagonists & inhibitors / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D004789:Enzyme Activation; D006358:Hot Temperature; D006801:Humans; D009154:Mutation; D011489:Protein Denaturation; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012436:S-Adenosylmethionine / Q000378:metabolism*
D011994:Recombinant Proteins; D012436:S-Adenosylmethionine; D003541:Cystathionine beta-Synthase