Ribonuclease catalytic mechanism

Despite considerable biochemical work, high-resolution crystal structure determination of native RNase A and S, and some medium-resolution studies of RNase A-inhibitor complexes, a number of questions existed concerning the details of the catalytic mechanism and the role of specific amino acids. Study of the low-temperature kinetics and three-dimensional structures of the significant steps of the ribonuclease reaction was designed to address the following questions. 

Many enzymes use coenzymes to achieve the detailed transformations they catalyze but the enzyme proteins themselves also supply important elements of the catalysis. Enzyme proteins are the source of the entire catalytic effect when coenzymes are not involved. As one common process, acid and base groups in enzymes perform proton transfers that are critical to the catalytic mechanism. A particularly informative example is observed in the enzyme ribonuclease A, which catalyzes the cleavage of RNA (16). The catalytic process (Fig. 4) involves the imidazole ring of the amino acid histidine that removes the proton from the 2-hydroxyl of the ribose. A different protonated histidine transfers a proton to the RNA to promote the cleavage process. Studies with D2O-H2O mixtures established that the two proton transfers occur at the same time (17). 

Sun W, Pertzev A, Nicholson AW. Catalytic mechanism of Escherichia coli ribonuclease 111 kinetic and inhibitor evidence for the involvement of two magnesium ions in RNA phosphodiester hydrolysis. Nucleic Acids Res. 2005 33(3) 807-815. 

In recent years attention has focused on the role of intrinsic binding energy and entropic factors as major contributors to enzyme catalytic efficiency (Page and Jencks, 197l Jencks, 1975,1981). The ribonuclease mechanism conforms to expectations based on these ideas. In particular, distortion occurs to raise the ground state of the substrate in the S complex, and the bound substrate interacts with the enzyme in a manner such that the enzyme becomes complementary to the transition state of the reaction during the catalytic cycle. 

A relative of the kinases is adenylate cyclase, whose role in forming the allosteric effector 3, 5 -cyclic AMP (cAMP) was considered in Chapter 11. This enzyme catalyzes a displacement on Pa of ATP by the 3 -hydroxyl group of its ribose ring (see Eq. 11-8, step a). The structure of the active site is known.905 Studies with ATPaS suggest an in-line mechanism resembling that of ribonuclease (step a, Eq. 12-25). However, it is Mg2+ dependent, does not utilize the two-histidine mechanism of ribonuclease A, and involves an aspartate carboxylate as catalytic base.906 All isoforms of adenylate cyclase are activated by the a subunits of some G proteins (Chapter 11). The structures907 of Gsa and of its complex with adenylate kinase905 have been determined. The Gsa activator appears to serve as an allosteric effector. 

The above-mentioned mechanism suggests that positioning the two histidines appropriately would lead to artificial ribonuclease under optimized pH conditions. Figure 6.13 shows an example of an artificial ribonuclease created in this way, which has a cyclodextrin core as the hydrophobic pocket and two histidine residues as catalytic sites. This artificial enzyme catalyzed the second step of the phosphodiester cleavage. The hydrophobic part of the cyclic phosphodiester (substrate) was accommodated into the core of the cyclodextrin and the phosphodiester was exposed between the two histidines. The water molecule was activated through proton removal (performed by the neutral histidine, left), and the activated water performed a nucleophilic attack on the phosphate atom. The protonated histidine (right) assisted this nucleophilic attack by protonating of the phosphodiester. Because of the cooperation between... 

The mechanism of ribonuclease A (RNase A) activity has been widely studied by evaluating different aspects such as roles of the catalytic amino acids, different substrates, thio effects, organic solvents, and pH and temperature dependence [79]. The usually accepted mechanism is the general acid/base pathway where a... 

Mechanistic studies are extremely valuable in guiding and understanding the catalyses possible with novel catalytic structures. Our study of the preferred mechanism for cleavage of RNA catalyzed by imidazole buffer revealed a detailed mechanism that is not related to the mechanism normally invoked for catalysis by the imidazole groups of ribonuclease A. Based on our studies, we have proposed a novel mechanism for ribonuclease A that is in good agreement with detailed evidence on the enzyme and with subsequent calculations. 

The Use of a Proton inventory to Explore the Mechanism of Ribonuclease Catalysis 440 A Substituent Effect Study to Decipher the Reason for the High Stability of Collagen 444 Using a Hammett Plot to Explore the Behavior of a Catalytic Antibody 450... 

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