Chen Y Q, Kraut J, Callender R
Department of Physics, City College of City University of New York, New York 10031, USA.
Biophys J. 1997 Feb;72(2 Pt 1):936-41. doi: 10.1016/s0006-3495(97)78727-7.
The catalytic site of all dihydrofolate reductases contains an invariant carboxylic acid, equivalent to Asp-27 in Escherichia coli dihydrofolate reductase (ecDHFR). It has been found that various kinetic and ligand binding properties of ecDHFR show a pH profile with a pKa of about 6.5. The group responsible for this pKa is often assumed to be carboxyl group of Asp-27. To determine the ionization state of this carboxyl and its pKa, we have employed a novel method, based on Raman difference spectroscopy, to obtain its vibrational spectrum in situ. The method is general for the study of protein carboxyl groups, which are often significantly implicated in protein function and structure; this study establishes the method's limits and problems. The Raman difference spectrum between wild-type ecDHFR and the Asp-27 to serine mutant (D27S) in the pH range 5.6-9.0 has been taken. No protonation of the carboxyl group was detected, implying that its pKa is probably less than 5.0. We did, however, detect a pH dependence in the intensity of Raman bands in the difference spectrum with a pKa of 6.3, indicating that the apo enzyme undergoes a pH-dependent conformational change. Because the carboxyl group of Asp-27 at the active site is the only ionizable group in the binding site, other groups, away from the catalytic site, must be responsible for the pH behavior of ecDHFR.
所有二氢叶酸还原酶的催化位点都含有一个不变的羧酸，相当于大肠杆菌二氢叶酸还原酶（ecDHFR）中的天冬氨酸-27。已发现ecDHFR的各种动力学和配体结合特性呈现出一个pKa约为6.5的pH谱。通常认为导致该pKa的基团是天冬氨酸-27的羧基。为了确定该羧基的电离状态及其pKa，我们采用了一种基于拉曼差光谱的新方法，以原位获得其振动光谱。该方法对于研究蛋白质羧基具有通用性，而蛋白质羧基在蛋白质功能和结构中常常起着重要作用；本研究确定了该方法的局限性和问题。我们获取了野生型ecDHFR与天冬氨酸-27突变为丝氨酸的突变体（D27S）在pH范围5.6 - 9.0内的拉曼差光谱。未检测到羧基的质子化，这意味着其pKa可能小于5.0。然而，我们确实在差光谱中检测到拉曼谱带强度存在pH依赖性，其pKa为6.3，这表明脱辅基酶经历了pH依赖性的构象变化。由于活性位点上天冬氨酸-27的羧基是结合位点中唯一可电离的基团，那么远离催化位点的其他基团必定是ecDHFR呈现pH行为的原因。
