Ribonuclease transitions

Navon A, Ittah V, Laity JH, et al. Local and long-range interactions in the thermal unfolding transition of bovine pancreatic ribonuclease A. Biochemistry 2001 40 93-104. 

Vanadium is beneficial and possibly essential for humans. It is certainly essential for a number of organisms. Vanadate (oxidation state V) and its derivatives are phosphate analogues, showing both ground state and transition state analogy (both structural and electronic) with phosphorus compounds. The analogy of five-coordinate vanadium compounds with the transition state of phosphate ester hydrolysis is well documented, and explains why so many vanadium compounds are potent inhibitors of phosphatases, ribonucleases and ATPases. 

Privalov, P.L., N.N. Khechinashvili, and B.P. Atanasov. 1971. Thermodynamic analysis of thermal transitions in globular proteins. I. Calorimetric study of chy-motrypsinogen, ribonuclease and myoglobin. Biopolymers 10 1865-1890. 

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. 

No large conformational changes occur in the enzyme during catalysis, but many small movements take place. The structural basis for the catalytic power of ribonuclease thus resides in several different features tight, specihc binding of a strained conformation of the substrate, general acid-base catalysis by His-12 and His-119, and preferential stabilization of the transition state by ionic interactions with Lys-41. 

The preceding summary and Fig. 20 present a frame-by-frame account of the pathway for ribonuclease catalysis, based predominandy on knowledge of the structures of the various intermediates and transition states involved. The ability to carry out such a study is dependent on three critical features (1) crystals of the enzyme which diffract sufficiently well to permit structural resolution to at least 2 A (2) compatibility of the enzyme, its crystals, and its catalytic kinetic parameters with cryoenzymology so as to permit the accumulation and stabilization of enzyme-substrate complexes and intermediates at subzero temperatures in fluid cryosolvents with crystalline enzyme and (3) the availability of suitable transition state analogs to mimic the actual transition states which are, of course, inaccessible due to their very short lifetimes. The results from this investigation demonstrate that this approach is feasible and can provide unparalleled information about an enzyme at work. 

Deng et al. (1993,1998) and Ray et al. (1993) have used V " as an analogue of in an attempt to model the transition state of the hydrolysis of phospho-diesters by ribonuclease A since is assumed to adopt the expected five coordination more readily than Examination of the vibrational spectrum of the vanadate analogue indicates that the terminal V-O bonds are only slightly weakened when bonded to the protein. A quantitative bond valence analysis effectively rules out two proposed mechanisms that involve the protonation of the terminal O atoms. 

Recently the related cyclization of the phenyl ester of c/j-tetrahydrofuran-3,4-diol monophosphate to the corresponding five-membered phosphate with loss of phenol has been shown to be subject to general catalysis by imidazole132. This reaction serves as a model for the first step in the action of ribonuclease which leads to the formation of the nucleoside 2 ,3 -cyclic phosphate. The actual details of the transition state leading to the cyclic phosphate as catalyzed by the enzyme are presently the subject of some debate. One possibility is the in-line mechanism (53)... 

Although inversion was not observed with the E. colt alkaline phosphatase, it has been observed for ribonucleases and many other hydrolytic enzymes and for most kinases transferring phospho groups from ATP. The difference lies in the existence of a phospho-enzyme intermediate in the action of alkaline phosphatase (see Eq. 12-38). Each of the two phosphotransferase steps in the phosphatase action apparently occurs with inversion. The simplest interpretation of all the experimental results is that phosphotransferases usually act by in-line -like mechanisms which may involve metaphosphate-ion-like transition states that are constrained to react with an incoming nucleophile to give inversion. An adjacent attack with pseudorotation would probably retain the original configuration and is therefore excluded. 

The enzyme consists of a single polypeptide chain of Mr 13 680 and 124 amino acid residues.187,188 The bond between Ala-20 and Ser-21 may be cleaved by subtilisin. Interestingly, the peptide remains attached to the rest of the protein by noncovalent bonds. The modified protein, called ribonuclease S, and the native protein, now termed ribonuclease A, have identical catalytic activities. Because of its small size, its availability, and its ruggedness, ribonuclease is very amenable to physical and chemical study. It was the first enzyme to be sequenced.187 The crystal structures of both forms of the enzyme were solved at 2.0-A resolution several years ago.189,190 Subsequently, crystal structures of many complexes of the enzyme with substrate and transition analogues and products have been solved at very high resolution.191 Further, because the catalytic activity depends on the ionizations of two histidine residues, the enzyme has been extensively studied by NMR (the imidazole rings of histidines are easily studied by this method—see Chapter 5). 

The methyl ester was crystallized and its absolute stereochemistry was determined by x-ray diffraction to be as in equation 8.37. This product corresponds to an in-line attack. When incubated with ribonuclease in aqueous solution, the methyl ester re-forms the original cyclic phosphorothioate (structure 8.36). This result is expected from the principle of microscopic reversibility, since the forward and reverse reactions must go through the same transition state. But it does show directly that the cyclization step involves an in-line attack an adjacent attack of the ribose hydroxyl in the cyclization of the methyl ester as in the right-hand structure 8.38 would give the enantiomer of structure 8.36. 

The fluorescence of ribonuclease solutions has been studied extensively by Cowgill. The absence of tryptophan permits the tyrosine fluorescence to be observed. The tyrosine fluorescence of RNase is very low in comparison with the maximum expected from its tyrosine content. All methods of denaturing RNase lead to an increase in fluorescence. Transitions, as indicated by the pattern of fluorescence change vs. denaturant concentration, are about the same as those indicated by other physical techniques [see, e.g., Gaily and Edelman (305) ]. 

Ribonuclease-A is inactive (C>p) in the presence of 2 mM SDS 308). The midpoint of the transition at pH 5.8 in 0.02 M phosphate buffer, 27°, as detected by fluorescence occurs at about 1 mM SDS. There is no additional acid transition detectable by fluorescence in spite of the fact that the SDS transition is supposed to normalize one residue while the acid transition affects two. 

Fig. 32. Transition T of 0.5% ribonuclease as a function of the concentration of added salts. pH = 7 (von Hippel and Wong222))...

Prutz WA, Butler J, Land EJ (1985a) Methionyl —> tyrosyl radical transitions initiated by Br2 in peptide model systems and ribonuclease A. Int J Radiat Biol 47 149-156 Prutz WA, Monig H, Butler J, Land E (1985b) Reactions of nitrogen dioxide in aqueous model systems oxidation of tyrosine units in peptides and proteins. Arch Biochem Biophys 243 125-134 Redpath JL, Willson RL (1975) Chain reactions and radiosensitization model enzyme studies. Int J Radiat Biol 27 389-398... 

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