Frank Nina, Kery Vladimir, Maclean Kenneth N, Kraus Jan P
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.
Biochemistry. 2006 Sep 12;45(36):11021-9. doi: 10.1021/bi060737m.
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.
胱硫醚β-合酶（CBS）是一种四聚体血红素蛋白，催化丝氨酸和同型半胱氨酸在磷酸吡哆醛依赖下缩合生成胱硫醚。CBS在甲硫氨酸循环和转硫途径之间占据关键的调节位置。人类CBS含有11个半胱氨酸残基，这些残基在哺乳动物中高度保守，但在催化相同反应的酵母酶中完全不存在，这表明其中一些残基可能具有调节作用。在本报告中，我们证明，无论是否存在CBS变构调节剂S-腺苷-L-甲硫氨酸（AdoMet），人类CBS中只有C15和C431可与溶剂接触。将C15突变为丝氨酸不影响催化作用或AdoMet激活，但显著降低了纯化酶在体外的聚集。C431的诱变导致CBS的一种组成型激活形式，其不能被AdoMet或热激活进一步激活。我们和其他人之前报道了一些C末端CBS点突变，这些突变导致对AdoMet的反应降低或消失。与所有这些之前研究的CBS突变体不同，CBS的C431突变形式无法结合AdoMet，这表明该残基要么直接参与AdoMet结合，要么其缺失诱导了一种构象变化，破坏了这种调节配体结合位点的完整性。
