Thompson J E, Raines R T
J Am Chem Soc. 1994 Jun;116(12):5467-8. doi: 10.1021/ja00091a060.
Bovine pancreatic ribonuclease A (RNase A) is a much studied enzyme that efficiently catalyzes the cleavage of RNA. The active site of RNase A contains two histidine residues with imidazole groups positioned to act as a general base (H12) and a general acid (H119) during catalysis of RNA cleavage. Recombinant DNA techniques were used to produce mutant enzymes in which either H12 or H119 was replaced with an alanine residue. Each mutation resulted in a 10-fold decrease in the value of / for cleaving either poly(C) or UpA. Thus, H12 and H119 each lower by 5–6 kcal/mol the free energy of the rate-limiting transition state during RNA cleavage. The value of / for cleavage of UpOCH--NO was decreased by 10-fold by replacing H12 but was unaffected by replacing H119. This result provides the first direct evidence that H119 acts as a general acid during catalysis by RNase A.
牛胰核糖核酸酶A(RNase A)是一种经过大量研究的酶,它能高效催化RNA的切割。RNase A的活性位点包含两个组氨酸残基,其咪唑基团在RNA切割催化过程中分别作为广义碱(H12)和广义酸(H119)发挥作用。利用重组DNA技术制备了突变酶,其中H12或H119被丙氨酸残基取代。每种突变导致切割聚(C)或UpA的kcat/Km值降低10倍。因此,H12和H119在RNA切割过程中各自将限速过渡态的自由能降低了5 - 6千卡/摩尔。通过替换H12,UpOCH₂NO的切割kcat/Km值降低了10倍,但替换H119对其没有影响。这一结果首次直接证明了H119在RNase A催化过程中作为广义酸发挥作用。