Steyaert J, Hallenga K, Wyns L, Stanssens P
Plant Genetic Systems NV, Gent, Belgium.
Biochemistry. 1990 Sep 25;29(38):9064-72. doi: 10.1021/bi00490a025.
Mechanisms for the ribonuclease T1 (RNase T1; EC 3.1.27.3) catalyzed transesterification reaction generally include the proposal that Glu58 and His92 provide general base and general acid assistance, respectively [Heinemann, U., & Saenger, W. (1982) Nature (London) 299, 27-31]. This view was recently challenged by the observation that mutants substituted at position 58 retain high residual activity; a revised mechanism was proposed in which His40, and not Glu58, is engaged in catalysis as general base [Nishikawa, S., Morioka, H., Kim, H., Fuchimura, K., Tanaka, T., Uesugi, S., Hakoshima, T., Tomita, K., Ohtsuka, E., & Ikehara, M. (1987) Biochemistry 26, 8620-8624]. To clarify the functional roles of His40, Glu58, and His92, we analyzed the consequences of several amino acid substitutions (His40Ala, His40Lys, His40Asp, Glu58Ala, Glu58Gln, and His92Gln) on the kinetics of GpC transesterification. The dominant effect of all mutations is on Kcat, implicating His40, Glu58, and His92 in catalysis rather than in substrate binding. Plots of log (Kcat/Km) vs pH for wild-type, His40Lys, and Glu58Ala RNase T1, together with the NMR-determined pKa values of the histidines of these enzymes, strongly support the view that Glu58-His92 acts as the base-acid couple. The curves also show that His40 is required in its protonated form for optimal activity of wild-type enzyme. We propose that the charged His40 participates in electrostatic stabilization of the transition state; the magnitude of the catalytic defect (a factor of 2000) from the His40 to Ala replacement suggests that electrostatic catalysis contributes considerably to the overall rate acceleration. For Glu58Ala RNase T1, the pH dependence of the catalytic parameters suggests an altered mechanism in which His40 and His92 act as base and acid catalyst, respectively. The ability of His40 to adopt the function of general base must account for the significant activity remaining in Glu58-mutated enzymes.
核糖核酸酶T1（RNase T1；EC 3.1.27.3）催化的酯交换反应机制通常包括以下观点：Glu58和His92分别提供广义碱和广义酸辅助作用[海涅曼，U.，& 森格尔，W.（1982年）《自然》（伦敦）299, 27 - 31]。最近这一观点受到了挑战，因为观察到在58位取代的突变体仍保留高残余活性；于是提出了一种修正机制，其中His40而非Glu58作为广义碱参与催化作用[西川，S.，森冈，H.，金，H.，富村，K.，田中，T.，上杉，S.，迫间岛，T.，富田，K.，大冢，E.，& 池原，M.（1987年）《生物化学》26, 8620 - 8624]。为阐明His40、Glu58和His92的功能作用，我们分析了几种氨基酸取代（His40Ala、His40Lys、His40Asp、Glu58Ala、Glu58Gln和His92Gln）对GpC酯交换动力学的影响。所有突变的主要影响在于催化常数（Kcat），这表明His40、Glu58和His92参与催化而非底物结合。野生型、His40Lys和Glu58Ala RNase T1的log（Kcat/Km）对pH的曲线，连同这些酶中组氨酸经核磁共振测定的pKa值，有力地支持了Glu58 - His92作为酸碱对起作用的观点。这些曲线还表明，野生型酶要达到最佳活性，His40需要以质子化形式存在。我们提出带电荷的His40参与过渡态的静电稳定作用；His40被Ala取代导致的催化缺陷程度（2000倍）表明，静电催化对总体速率加速有很大贡献。对于Glu58Ala RNase T1，催化参数的pH依赖性表明其机制发生了改变，其中His40和His92分别作为碱和酸催化剂起作用。His40能够发挥广义碱的功能，这必然解释了Glu58突变酶中仍保留的显著活性。
