Department of Biological Chemistry, University of Michigan Medical Center, Ann Arbor, Michigan 48109-0600, USA.
J Biol Chem. 2012 Dec 21;287(52):43464-71. doi: 10.1074/jbc.M112.414722. Epub 2012 Nov 2.
Human cystathionine β-synthase (CBS), a novel heme-containing pyridoxal 5'-phosphate enzyme, catalyzes the condensation of homocysteine and serine or cysteine to produce cystathionine and H(2)O or H(2)S, respectively. The presence of heme in CBS has limited spectrophotometric characterization of reaction intermediates by masking the absorption of the pyridoxal 5'-phosphate cofactor. In this study, we employed difference stopped-flow spectroscopy to characterize reaction intermediates formed under catalytic turnover conditions. The reactions of L-serine and L-cysteine with CBS resulted in the formation of a common aminoacrylate intermediate (k(obs) = 0.96 ± 0.02 and 0.38 ± 0.01 mM(-1) s(-1), respectively, at 24 °C) with concomitant loss of H(2)O and H(2)S and without detectable accumulation of the external aldimine or other intermediates. Homocysteine reacted with the aminoacrylate intermediate with k(obs) = 40.6 ± 3.8 s(-1) and re-formed the internal aldimine. In the reverse direction, CBS reacted with cystathionine, forming the aminoacrylate intermediate with k(obs) = 0.38 ± 0.01 mM(-1) s(-1). This study provides the first insights into the pre-steady-state kinetic mechanism of human CBS and indicates that the reaction is likely to be limited by a conformational change leading to product release.
人胱硫醚β-合酶（CBS），一种新型含血红素的吡哆醛 5'-磷酸酶，催化同型半胱氨酸与丝氨酸或半胱氨酸缩合，分别生成胱硫醚和 H₂O 或 H₂S。CBS 中的血红素限制了通过掩盖吡哆醛 5'-磷酸辅因子的吸收对反应中间产物的分光光度法特征。在这项研究中，我们采用差示停流光谱法在催化周转条件下对形成的反应中间体进行了表征。L-丝氨酸和 L-半胱氨酸与 CBS 的反应形成了一个共同的氨基丙烯酸中间产物（k(obs) = 0.96 ± 0.02 和 0.38 ± 0.01 mM(-1) s(-1)，分别在 24°C 下），同时伴随着 H₂O 和 H₂S 的损失，并且没有检测到外部亚胺或其他中间产物的积累。同型半胱氨酸与氨基丙烯酸中间产物反应的 k(obs) = 40.6 ± 3.8 s(-1)，重新形成内部亚胺。在相反方向上，CBS 与胱硫醚反应，形成氨基丙烯酸中间产物，k(obs) = 0.38 ± 0.01 mM(-1) s(-1)。这项研究首次深入了解了人 CBS 的预稳态动力学机制，并表明该反应可能受到导致产物释放的构象变化的限制。
