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.
Department of Cellular & Structural Biology, University of Colorado School of Medicine, Denver, Colorado, 80262, USA.
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Kraus
D000595:Amino Acid Sequence; D001665:Binding Sites; D017124:Conserved Sequence; D003541:Cystathionine beta-Synthase / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D004789:Enzyme Activation / Q000187:drug effects; D006418:Heme / Q000378:metabolism; D006358:Hot Temperature; D006801:Humans; D006868:Hydrolysis; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010446:Peptide Fragments / Q000378:metabolism; D011489:Protein Denaturation; D011732:Pyridoxal Phosphate / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D012436:S-Adenosylmethionine / Q000494:pharmacology; D012997:Solvents; D013050:Spectrometry, Fluorescence; D014357:Trypsin / Q000378:metabolism*; D014364:Tryptophan / Q000378:metabolism
D010446:Peptide Fragments; D011994:Recombinant Proteins; D012997:Solvents; D006418:Heme; D011732:Pyridoxal Phosphate; D012436:S-Adenosylmethionine; D014364:Tryptophan; D014357:Trypsin; D003541:Cystathionine beta-Synthase