Etoh Y, Shoun H, Ogino T, Fujiwara S, Arima K, Beppu T
J Biochem. 1982 Jun;91(6):2039-46. doi: 10.1093/oxfordjournals.jbchem.a133897.
The location and state of an essential histidyl residue in a milk-clotting acid proteases, Mucor rennin, were investigated by NMR spectroscopy. Assignment of the C2H resonance peak of the essential histidyl residue was possible by comparison of the NMR spectrum of the native enzyme with that of the photo-oxidized enzyme. The pH titration curve for the chemical shift of the C2H proton showed two inflections, a major one with pKa = 7.4 and a minor one with pKa = 3.5 at 30 degrees C. The major inflection, corresponding to an intrinsic protonation of the imidazole ring, shifted toward the alkaline side upon addition of acetyl pepstatin, an inhibitor specific for the acid protease. Modification of an essential carboxyl group in the enzyme with diazoacetyl-DL-norleucine caused disappearance of the minor inflection as well as an acidic shift of the major pKa value. Perturbation effects on the C2H resonance of the lanthanide metals, Pr3+, Eu3+, and Gd3+, suggested their selective binding to a carboxyl group and location of the bound metal atom close to the essential histidyl residue. All data suggested that the essential histidyl residue of Mucor rennin is located close to one of the two essential carboxyl groups in the catalytic site of the enzyme.
通过核磁共振光谱法研究了凝乳酸性蛋白酶毛霉凝乳酶中一个必需组氨酸残基的位置和状态。通过将天然酶的核磁共振光谱与光氧化酶的光谱进行比较，可以确定必需组氨酸残基的C2H共振峰。C2H质子化学位移的pH滴定曲线在30℃时显示出两个拐点，一个主要拐点的pKa = 7.4，一个次要拐点的pKa = 3.5。对应于咪唑环固有质子化的主要拐点，在加入酸性蛋白酶特异性抑制剂乙酰胃蛋白酶抑制剂后向碱性侧移动。用重氮乙酰-DL-正亮氨酸修饰酶中的一个必需羧基，导致次要拐点消失以及主要pKa值向酸性方向移动。镧系金属Pr3+、Eu3+和Gd3+对C2H共振的扰动效应表明它们选择性地结合到一个羧基上，并且结合的金属原子位于靠近必需组氨酸残基的位置。所有数据表明，毛霉凝乳酶的必需组氨酸残基位于酶催化位点中两个必需羧基之一附近。
