Seryl hydroxyl groups

In the reaction with PNPA, myristoylhistidine [29] in a cationic micelle rapidly forms acetylimidazole as a fairly stable intermediate which is readily observable at 245 nm. On the other hand, a mixed micelle of [29] and N,N-dimethyl-N-2-hydroxyethylstearylammonium bromide [30] leads to the formation and decay of the intermediate, indicating that the acetyl group is transferred from imidazole to hydroxyl groups (Tagaki et al., 1977 Tagaki et al., 1979). This can be a model of cr-chymotrypsin which catalyses hydrolysis of PNPA (non-specific substrate) by initial acylation of the histidyl imidazole followed by acyl transfer to the seryl hydroxyl group (Kirsh and Hubbard, 1972), as indicated schematically in (12). 

A charge relay system (Blow, 1976) exists in a number of hydrolytic enzymes. In such systems, an aspartate carboxylate group buried in a hydrophobic microenvironment activates a seryl hydroxyl group through hydrogen bonding. Thus, it is interesting to study the effect of carboxylate ions on other nucleophiles in aprotic media. 

In die above examples, the concerted acticm of the two functional groups is not observed. In contrast, the seryl hydroxyl group and the histidyl imidazole group act upon die substrate in a concerted manner in the charge relay system (see Ffg. 2-1). Concerted bifunctional catalysis was observed when a binary copolymer ctHitmning... 

Enzymes with seryl hydroxyl groups at the active sites can be inactivated by organophosphorous compounds. Thus, diisopropylphosphofluoridate (DPF) inactivates serine hydrolases by phosphorylation at the active site ... 

It must be pointed out, however, that the catalytic role played by the imidazole group at the active site of serine esterases is different from tlmt of Eq. (4—1). The imidazole group at the active site helps acylation and deacylation at the seryl hydroxyl group as ageneralbase (see Fig. 2—1), whereas in Eq. (4—1) imidazole acts as a nucleophilic catalyst. 

FIGURE 6.5 Transfer of acyl between the amino and hydroxyl groups of seryl. (A) Deprotonation of O-acylseryl- induces oxazolidine formation, which is followed by (B) rearrangement to A-acylseryl-. (C) Protonation of the carbonyl of A-acylseryl- by mineral acid results in dehydration to the oxazoline, which is followed by hydrolysis (D) at the double bond giving protonated O-acylseryl-. 

This group includes the chymotrypsins, trypsin, elastase, thrombin, and subtilisin. The name of this group of enzymes refers to the seryl residue that is involved in the active site. As a consequence, all of these enzymes are inhibited by diisopropylphosphorofluori-date, which reacts with the hydroxyl group of the seryl residue. They also have an imidazole group as part of the active site and they are all endopeptides. The chymotrypsins, trypsin and elastase, are pancreatic enzymes that carry out their function in the intestinal... 

Both steps of the process are catalyzed by the basic form of the imidazole group of a histidine residue forming part of the active site. If the native conformation of the enzyme is disrupted by denaturation reagents such as urea, the unique seryl hydroxyl loses its characteristic reactivity in both the acylation and the deacylation process (15). This is easily understood if we realize that the reactive serine and the catalytically active histidine are extremely far from one another along the polypeptide chain, being separated by 137 amino acid residues (16) they are brought into the necessary juxtaposition only by the specific folding in the native enzyme structure. The mechanism by which the enzyme acetylcholine esterase catalyzes the hydrolysis of its substrate acetylcholine appears to be very similar (17). As we shall see, a number... 

Chymotrypm, probably the most thorou y studied enzyme because of its stability and availability, primarily catalyzes the hydrolysis of amide bonds of proteins and peptides adjacent to the carbonyl group of the aromatic L-amino acid residues of tryptophan, tyro e, and phenylalanine. Therefore, the hydrophobic interaction between the active site and substrate molecules is believed to make a mryor contribution to the stabflity of enzyme-substrate complexes. In fact, the X-ray data showed that the a-chymotryfsin molecule is an elipsoid (51x40x40 A) and that the active site is a hydrophobic cavity of 10—12 Ax5.5 —6.5 Ax3.5 —4.5 A (5). In chemical modification experiments, histidyl imidazole 2, and seryl hydroxyl 1 groups were found to be directly involved in the catal) ic process. The formation of acyl enzyme intermediates at the seryl residue was demonstrated by phyacal and chemical means. 

Figure 1.9 Mucin-type Sequences of Glycoproteins. These structures share the common feature of N-acetylgalactosamine a-linked to the hydroxyl groups of seryl or threonyl residues otherwise they are extremely variable. Structures (a) to (d) occur in rat colonic mucus (Slomiany, Murty and Slomiany, 1980) (e) and (f) are of widespread occurrence and (g) is known from human gastric mucin and forms the inner part of several larger saccharides from human and porcine sources. Structure (a) is also very commonly found.

An enzyme reaction intermediate (Enz—O—C(0)R or Enz—S—C(O)R), formed by a carboxyl group transfer (e.g., from a peptide bond or ester) to a hydroxyl or thiol group of an active-site amino acyl residue of the enzyme. Such intermediates are formed in reactions catalyzed by serine proteases transglutaminase, and formylglyci-namide ribonucleotide amidotransferase . Acyl-enzyme intermediates often can be isolated at low temperatures, low pH, or a combination of both. For acyl-seryl derivatives, deacylation at a pH value of 2 is about 10 -fold slower than at the optimal pH. A primary isotope effect can frequently be observed with a C-labeled substrate. If an amide substrate is used, it is possible that a secondary isotope effect may be observed as welF. See also Active Site Titration Serpins (Inhibitory Mechanism)... 

Displacement of the OH group of seryl-tRNA by an -SeH group to yield selenocysteyl-tRNA requires the activation of the hydroxyl. When the selA gene product was purified, it was found that the protein contains pyridoxal phosphate as a prosthetic group. Accordingly, the reaction mechanism involves the formation of a Schiff base between the carbonyl of the pyridoxal moiety and the o -amino group... 

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