Südi J
Biochem J. 1974 Apr;139(1):251-9. doi: 10.1042/bj1390251.
The stopped-flow kinetic studies described in this and the following paper (Südi, 1974) demonstrate that a Haldane-type description of the reversible lactate dehydrogenase reaction presents an experimentally feasible task. Combined results of these two papers yield numerical values for the six rate constants defined by the following equilibrium scheme, where E represents lactate dehydrogenase: [Formula: see text] The experiments were carried out at pH8.4 at a relatively low temperature (6.3 degrees C) with the pig heart enzyme. Identification of the above two intermediates and determination of the corresponding rate constants actually involve four series of independent observations in these studies, since (a) the reaction can be followed in both directions, and (b) both the u.v. absorption and the fluorescence of the coenzymes are altered in the reaction, and it is shown that these two spectral changes do not occur simultaneously. Kinetic observations made in the reverse direction are reported in this paper. It is demonstrated that the fluorescence of NADH can no longer be observed in the ternary complex E(NADH) (Pyr). Even though the oxidation-reduction reaction rapidly follows the formation of this complex, the numerical values of k(-4) (8.33x10(5)m(-1).s(-1)) and k(+4) (222s(-1)) are easily obtained from a directly observed second-order reaction step in which fluorescent but not u.v.-absorbing material is disappearing. U.v.-absorption measurements do not clearly resolve the subsequent oxidation-reduction step from the dissociation of lactate. It is shown that this must be due partly to the instrumental dead time, and partly to a low transient concentration of E(NAD+) (Lac) in the two-step sequential reaction in which the detectable disappearance of u.v.-absorbing material takes place. It is estimated that about one-tenth of the total change in u.v. absorption is due to a ;burst reaction' in which E(NAD+) (Lac) is produced, and this estimation yields, from k(obs.)=120s(-1), k(-2)=1200s(-1).
本论文及后续论文（许迪，1974年）中描述的停流动力学研究表明，对可逆乳酸脱氢酶反应进行哈代型描述是一项实验上可行的任务。这两篇论文的综合结果得出了由以下平衡方案定义的六个速率常数的数值，其中E代表乳酸脱氢酶：[公式：见原文] 实验在pH8.4、相对低温（6.3摄氏度）下用猪心酶进行。在这些研究中，鉴定上述两种中间体并确定相应的速率常数实际上涉及四组独立观察，因为（a）反应可以双向跟踪，并且（b）辅酶的紫外吸收和荧光在反应中都会改变，并且表明这两种光谱变化不会同时发生。本文报道了反向进行的动力学观察结果。结果表明，在三元复合物E（NADH）（Pyr）中不再能观察到NADH的荧光。尽管氧化还原反应在该复合物形成后迅速发生，但k（-4）（8.33×10⁵m⁻¹·s⁻¹）和k（+4）（222s⁻¹）的数值很容易从一个直接观察到的二级反应步骤中获得，在该步骤中，有荧光但无紫外吸收的物质正在消失。紫外吸收测量无法清楚地将随后的氧化还原步骤与乳酸的解离区分开。结果表明，这部分是由于仪器的死时间，部分是由于在两步连续反应中E（NAD⁺）（Lac）的瞬态浓度较低，在该反应中发生了可检测到的紫外吸收物质的消失。据估计，紫外吸收的总变化中约十分之一是由于产生E（NAD⁺）（Lac）的“爆发反应”，并且从k（obs.）=120s⁻¹得出该估计值，k（-2）=1200s⁻¹。
