Ribonuclease, conformation mechanism

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. 

For ribonuclease A the occurrence of conformational changes and the occurrence of acid-base catalysis has been well documented. The overall mechanism can be envisaged as follows. The enzyme exists in dynamic equilibrium between two forms differing in the structure of the active site groove. The substrate is bound almost as rapidly as it can diffuse to the active site. Binding of the substrate induces a conformational change that... 

This situation was presaged by Westheimer (1980), who stated ... that all enzymic reactions at phosphorus proceed with inversion and therefore they occur without pseudorotation at phosphorus. The argument for such a statement is based on two tenets first, the molecular motion caused by pseudorotation may require a conformational change of the enzyme to accommodate this movement secondly, a pseudorotation pathway will require a multistep mechanism. For example, the cyclization step of the reaction catalysed by ribonuclease may be postulated to involve pseudo-... 

In the absence of substrates, a relaxation process is observed in solutions of ribonuclease with the temperature jump method having a relaxation time of 0.1 to 1 msec. This relaxation time is independent of the enzyme concentration but is pH dependent. It can be attributed to an isomerization or conformational change of the enzyme, and a simple mechanism consistent with the data is... 

Fig. 9-7 A schematic mechanism for the hydrolysis of a dinucleoside by ribonuclease A. The various conformational states inferred to be present from kinetic studies are shown. In this mechanism PypN is a pyrimidine 3 5 -nucleoside, Py 2 3 p is a pyrimidine 2 3 cyclic phosphate and E is the enzyme. The primes designate different conformational states of the enzyme and enzyme-substrate complexes.

Further examples of conformational adaptationf will be given, each one quite individual in its mechanism, yet collectively supplying a picture of considerable uniformity. Ribonuclease forms a compact structure, hydrophilic outside, lipophilic inside, with a slot to receive the substrate. The active site makes use of histidine residues (numbers 12 and 119) that would otherwise be remote from one another (Kartha, Bello and Marker, 1967). The dimensions of the folded ribonuclease molecule are about 30X 30X 38 A (mol. wt. 15 000). When charged with a substrate, e.g. cytidine phosphate, one histidine residue binds the phosphate group, the other the sugar. The tertiary structure of a-chymotrypsin has been similarly worked out the active site depends on the closeness of two amino acids that are on different strands, namely serine-195 and histidine-57... 

Most of the main concepts regarding the mechanisms of protein folding accepted today originated from both theoretical conformational computations and the determination of structures at atomic resolution. The amount of experimental data is still insufficient to allow a high degree of generalization. The number of known proteins for which a detailed and complete study of the folding process is available, remains indeed very small. Well documented systems such as ribonuclease, staphylococcal nuclease, BPTI, lysozyme, serine proteases, and few other proteins are used frequently as examples in the discussion that follows. 

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