Biochemistry - The Chemical Reactions of Living Cells, Volume 2 - D. Metzler 1980

Types of Reactions Catalyzed by Enzymes
Substitution at the Phosphorus Atom
Ribonuclease

In 1972, the Nobel Prize in Chemistry was awarded to Stanford Moore, William H. Stein, and Christian B. Anfinsen for their studies on ribonuclease, a digestive enzyme secreted by the Pancreas responsible for RNA Cleavage. Stein and Moore, who had previously developed ion-exchange Methods for separating Amino Acids and Peptides, determined the Amino Acid Sequence of the bovine pancreatic enzyme, which consists of 124 amino acid residues [57]. Before X-ray crystallographic studies were initiated, it was established that Lys-41 exhibits unusually high reactivity toward dinitrofluorobenzene. Furthermore, based on photooxidation inactivation data, Stein and Moore concluded that two Histidine residues, His-12 and His-119, located near nearly opposite ends of the polypeptide chain, are Components of the Active Site. The results of crystallographic studies demonstrate that Lys-41 and the two histidine residues are indeed located close to one another within the cleft containing the active site [58].

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The catalytic cleavage of phosphodiester bonds in the RNA molecule by ribonuclease proceeds in two stages. The adjacent hydroxyl group at the 2' position of the ribose ring is apparently deprotonated via attack by base B (stage a in equation (7-19)).

The resulting alkoxide ion then attacks the phosphorus atom, displacing the oxygen linked to the 5'-carbon of the neighboring nucleotide unit. Subsequent cleavage can be facilitated by proton transfer from the acidic group B'H⁺. A cyclic 2',3'-diphosphate is formed as an intermediate, which is then hydrolyzed by a Water molecule in stage b to yield the free 3'-nucleotide. Thus, the overall reaction represents a two-stage double-displacement process, much like that observed with α-Chymotrypsin, with the difference that the nucleophilic catalyst is an adjacent group within the substrate molecule rather than an amino acid side chain.

The most valuable insight derived from X-Ray Diffraction data is that the primary group abstracting a proton from the 2'-hydroxyl is His-12, whereas the acidic group donating a proton to the departing 5'-oxygen belongs to His-119 [59]. (It is curious, however, that a synthesized ribonuclease derivative bearing an N^τ-carboxymethylated His-12 residue retains some catalytic activity—a fact that raises several questions [60].) The pH dependence profile of ribonuclease activity is consistent with the proposed mechanism, as two pK_a values (5.4 and 6.4) have been found corresponding to the two groups whose ionization state governs enzyme activity. (Based on the NMR spectra shown in Fig. 2-42, a pK_a value of 5.8 was obtained.) A Lys-41 residue is located in the vicinity of the two histidine residues. Its positive charge is presumably utilized to partially neutralize the negative charge on the phosphate oxygen atoms, thereby facilitating attack by the nucleophilic agent. From the perspective of ribonuclease chemistry, it is noteworthy that bacterial peptidase Treatment cleaves off a fragment containing twenty amino acid residues. This "S-peptide" can recombine with the remainder of the molecule to form an active enzyme designated as ribonuclease S. The Structure of this enzyme was determined by X-ray diffraction and was found to be essentially identical to that of native ribonuclease.

Protein Synthesis methods are now advanced to the point where small Enzymes can be synthesized in the laboratory. This makes it possible to generate novel modified enzymes and critically analyze the roles of various active-site groups. For instance, it has been established that a synthetic peptide consisting of 70 amino acid residues, analogous to ribonuclease S but bearing several deletions and lacking Disulfide Bonds entirely, nevertheless retains appreciable catalytic activity [61].