Ribonuclease covalent structure

Enzymatic activity, however, is not merely associated with covalent structures, but chiefly with tertiary structure which is still more difficult to determine. The crucial role of tertiary structure is proved by the fact that denaturation brings about inactivation. Even with proteins which may be reversibly denatured, such as chymotrypsin and trypsin, activity is lost as long as denaturation persists. Ribonuclease appeared for a while to be an exception, since it was still active in 8 M urea. But it was shown later that phosphate ions, at a concentration as low as 0.003 M, and polyphosphates induced in urea-denatured ribonuclease spectral changes usually associated with refolding (164). It could then be assumed that ribonucleic acid, the actual substrate, was also able to refold the denatured form and prevent inactivation in this way. In other words, even in ribonuclease, the active center is probably not built by adjacent residues in a tail or a ring, but by some residues correctly located in space by the superimposed... 

The complete covalent structure of ribonuclease, i.e., the amino acid sequence and disulfide bridge positions, has recently been determined (Hirs et al.y 1960 Spackman et al., 1960) and is shown in Fig. 154. The molecule consists of a single polypeptide chain of 124 amino acid residues with lysine and valine as the N-terminal and C-terminal groups, respectively (Anfinsen... 

Covalent bridging of biopolymers is one of the widely occurring prindples in nature for increasing the stability of the tertiary structure, for example the disulfide bridges in keratin and ribonuclease. 

Unfortunately, the size of the crystallographic problem presented by elastase coupled with the relatively short lifedme of the acyl-enzyme indicated that higher resolution X-ray data would be difficult to obtain without use of much lower temperatures or multidetector techniques to increase the rate of data acquisition. However, it was observed that the acyl-enzyme stability was a consequence of the known kinetic parameters for elastase action on ester substrates. Hydrolysis of esters by the enzyme involves both the formation and breakdown of the covalent intermediate, and even in alcohol-water mixtures at subzero temperatures the rate-limidng step is deacylation. It is this step which is most seriously affected by temperature, allowing the acyl-enzyme to accumulate relatively rapidly at — 55°C but to break down very slowly. Amide substrates display different kinetic behavior the slow step is acylation itself. It was predicted that use of a />-nitrophenyl amid substrate would give the structure of the pre-acyl-enzyme Michaelis complex or even the putadve tetrahedral intermediate (Alber et ai, 1976), but this experiment has not yet been carried out. Instead, over the following 7 years, attention shifted to the smaller enzyme bovine pancreatic ribonuclease A. 

In small proteins, hydrophobic residues are less likely to be sheltered in a hydrophobic interior—simple geometry dictates that the smaller the protein, the lower the ratio of volume to surface area. Small proteins also have fewer potential weak interactions available to stabilize them. This explains why many smaller proteins such as those in Figure 4—18 are stabilized by a number of covalent bonds. Lysozyme and ribonuclease, for example, have disulfide linkages, and the heme group in cytochrome c is covalently linked to the protein on two sides, providing significant stabilization of the entire protein structure. 

Vanadate itself is a much poorer inhibitor of ribonuclease A than is the VUr complex. This makes sense because vanadate alone cannot mimic the transition state. Covalently bound components, the parts of the nucleoside covalently bound to the phosphate moiety, are needed to complete the transition-state-like structure. They... 

Many proteins, including almost all of those that are secreted from cells and many that are components of cell surfaces, carry covalently attached oligosaccharides.These glycoproteins may carry just one or a few, often highly branched, oligosaccharide chains. For example, ribonuclease B has a structure identical to that of ribonuclease A (Fig. 12-25) except for the presence of an oligosaccharide on asparagine 34. ... 

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