Trypsin cleavage of human cystathionine beta-synthase into an evolutionarily conserved active core: structural and functional consequences.

Cystathionine beta-synthase (CBS) catalyzes the condensation of homocysteine and serine to cystathionine-an irreversible step in the eukaryotic transsulfuration pathway. The native enzyme is a homotetramer or multimer of 63-kDa (551 amino acids) subunits and is activated by S-adenosyl-l-methionine (AdoMet) or by partial cleavage with trypsin. Amino-terminal analysis of the early products of trypsinolysis demonstrated that the first cleavages occur at Lys 30, 36, and 39. The enzyme still retains the subunit organization as a tetramer or multimer composed of 58-kDa subunits. Analysis by electrospray ionization mass spectrometry showed that further trypsin treatment cleaves CBS in its COOH-terminal region at Arg 413 to yield 45-kDa subunits. This 45-kDa active core is the portion of CBS most conserved with the evolutionarily related enzymes isolated from plants, yeast, and bacteria. The active core of CBS forms a dimer of approximately 85 kDa. The dimer is about twice as active as the tetramer. It binds both pyridoxal 5'-phosphate and heme cofactors but is no longer activated by AdoMet. Further analysis suggests that the dissociation of CBS to dimers causes a decrease in enzyme thermostability and a threefold increase in affinity toward the sulfhydryl-containing substrate-homocysteine. We found that the COOH-terminal region, residues 414-551, is essential for maintaining the tetrameric structure and AdoMet activation of the enzyme. The inability of the active core to form multimeric aggregates has facilitated its crystallization and X-ray diffraction studies.