Sergienko E A, Wang J, Polovnikova L, Hasson M S, McLeish M J, Kenyon G L, Jordan F
Department of Chemistry, Rutgers, the State University, Newark, New Jersey 07102, USA.
Biochemistry. 2000 Nov 14;39(45):13862-9. doi: 10.1021/bi001214w.
Thiamin diphosphate (ThDP)-dependent enzymes catalyze a range of transformations, such as decarboxylation and ligation. We report a novel spectroscopic assay for detection of some of the ThDP-bound intermediates produced on benzoylformate decarboxylase. Benzoylformate decarboxylase was mixed with its alternate substrate p-nitrobenzoylformic acid on a rapid-scan stopped-flow instrument, resulting in formation of three absorbing species (lambda(max) in parentheses): I(1) (a transient at 620 nm), I(2) (a transient at 400 nm), and I(3) (a stable absorbance with lambda(max) > 730 nm). Analysis of the kinetics of the two transient species supports a model in which a noncovalent complex of the substrate and the enzyme is converted to the first covalent intermediate I(1); the absorbance corresponding to I(1) is probably a charge-transfer band arising from the interaction of the thiamin diphosphate-p-nitrobenzoylformic acid covalent adduct (2-p-nitromandelylThDP) and the enzyme. The rate of disappearance of I(1) parallels the rate of formation of I(2). Chemical models suggest the lambda(max) of I(2) (near 400 nm) to be appropriate to the enamine, a key intermediate in ThDP-dependent reactions resulting from the decarboxylation of the thiamin diphosphate-p-nitrobenzoylformic acid covalent adduct. Therefore, the rate of disappearance of I(1) and/or the appearance of I(2) directly measure the rate of decarboxylation. A relaxation kinetic treatment of the pre-steady-state kinetic data also revealed a hitherto unreported facet of the mechanism, alternating active-sites reactivity. Parallel studies of the His70Ala BFD active-site variant indicate that it cannot form the complex reported by the charge-transfer band (I(1)) at the level of the wild-type protein.
硫胺素二磷酸（ThDP）依赖性酶催化一系列转化反应，如脱羧反应和连接反应。我们报道了一种新型光谱测定法，用于检测苯甲酰甲酸脱羧酶产生的一些与ThDP结合的中间体。在快速扫描停流仪器上，将苯甲酰甲酸脱羧酶与其替代底物对硝基苯甲酰甲酸混合，产生了三种吸收物种（括号内为最大吸收波长）：I(1)（620 nm处的瞬态）、I(2)（400 nm处的瞬态）和I(3)（最大吸收波长>730 nm的稳定吸光度）。对这两种瞬态物种动力学的分析支持了一个模型，即底物与酶的非共价复合物转化为第一个共价中间体I(1)；对应于I(1)的吸光度可能是硫胺素二磷酸 - 对硝基苯甲酰甲酸共价加合物（2 - 对硝基扁桃基硫胺素二磷酸）与酶相互作用产生的电荷转移带。I(1)的消失速率与I(2)的形成速率平行。化学模型表明I(2)（接近400 nm）的最大吸收波长适合烯胺，烯胺是硫胺素二磷酸 - 对硝基苯甲酰甲酸共价加合物脱羧产生的ThDP依赖性反应中的关键中间体。因此，I(1)的消失速率和/或I(2)的出现直接测量脱羧速率。对稳态前动力学数据的弛豫动力学处理还揭示了该机制一个迄今未报道的方面，即交替活性位点反应性。对His70Ala BFD活性位点变体的平行研究表明，它不能在野生型蛋白水平形成由电荷转移带报道的复合物（I(1)）。
