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, rep resents a key regulatory point in the biosynthesis of cysteine via the tran ssulfuration pathway. Inherited deficiency of CBS causes homocystinuria. CB S is activated by S-adenosyl-L-methionine(AdoMet) by inducing a conformatio nal change involving a noncatalytic C-terminal region spanning residues 414 -551. We report the purification of two patient-derived C-terminal mutant f orms of CBS, S466L and I435T, that provide new insight into the mechanism o f CBS regulation and indicate a regulatory function for the "CBS domain". B oth of these point mutations confer catalytically active proteins. The I435 T 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 A doMet. Both mutant proteins are able to bind Adc,Met indicating that their impairment is related to their ability to assume the fully activated confor mation 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 W T, S466L, and a truncated form of CBS lacking the C-terminal region cannot be further activated by this treatment. Tryptophan and PLP fluorescence dat a for these different forms of CBS indicate that activation by AdoMet, limi ted proteolysis, and thermal denaturation share a common mechanism involvin g the displacement of an autoinhibitory domain located in the C-terminal re gion of the protein.