Peptide enzyme active sites

Nonrepetitive but well-defined structures of this type form many important features of enzyme active sites. In some cases, a particular arrangement of coil structure providing a specific type of functional site recurs in several functionally related proteins. The peptide loop that binds iron-sulfur clusters in both ferredoxin and high potential iron protein is one example. Another is the central loop portion of the E—F hand structure that binds a calcium ion in several calcium-binding proteins, including calmodulin, carp parvalbumin, troponin C, and the intestinal calcium-binding protein. This loop, shown in Figure 6.26, connects two short a-helices. The calcium ion nestles into the pocket formed by this structure. 

Peptide-Carbohydrate Mimicry in Enzyme Active Sites. 93... 

These hemiketalic adducts are very good mimics of the tetrahedral transition state involved in the enzymatic hydrolysis of an ester bond or a peptidic bond [71,72], The nucleophilic entity of the enzyme active site (e.g. the hydroxyl of hydrolytic serine enzymes) can easily add onto the activated carbonyl of a fluor-oketone leading to a very stable tetrahedral intermediate. The enzyme is not regenerated and is thus inhibited (Fig. 21) [73],... 

In the presence of PPi, known to bind strongly to the enzyme active site (Section III,E), there was a weak protective effect. The experimental points fell in the shaded area of Fig. 6, and the data were analyzed with equations developed by Scrutton and Utter (45). The results of this treatment led to the conclusion that TNBS can react with both free enzyme and enzyme-PPi complex to cause catalytic inactivation the differences are only quantitative (45). Either TNBS can displace PP, from an active site lysine or TNBS modifies a different lysine, apart from the active site, and the presence of PPi on the enzyme partially protects against TNBS inactivation by some indirect mechanism. Unfortunately, as discussed above, this issue cannot be settled with these kinetic analyses. Furthermore, because all of the enzyme lysines are to some extent reactive with TNBS (Fig. 5), the single super-reactive lysine whose modification leads to inactivation cannot be isolated and identified, as, for example, in a particular peptide fragment. A variety of interpretations are possible, as discussed elsewhere (45). 

However, an 18-residue segment of the autoinhibitory domain, composed of two short a-helical regions, was clearly visible in close contact with the catalytic domain, spanning the enzyme active site. This observation was consistent with previous studies showing competitive inhibition kinetics using a 25-residue peptide from the calcineurin A autoinhibitory domain (Parsons et al., 1994 Sagoo et al., 1996). 

In models for carboxypeptidase A we showed the intracomplex catalyzed hydrolysis of an ester by a metal ion and a carboxylate ion [106], which are the catalytic groups of carboxypeptidase A. Some mechanistic proposals for the action of carboxypeptidase involve an anhydride intermediate that then hydrolyzes to the product and the regenerated enzyme. Although we later found convincing evidence that the enzyme does not use the anhydride mechanism in cleaving peptides [96-99], it may well use such a mechanism with esters. In a mimic of part of this mechanism we showed [107], but see also Ref. 108, that we could achieve very rapid hydrolysis of an anhydride by bound Zn2+, which is the metal ion in the enzyme. In another model, a carboxylate ion and a phenolic hydroxyl group, which are in the enzyme active site, could cooperatively catalyze the cleavage of an amide by the anhydride mechanism [109]. 

The specificity of trypsin toward peptide bonds adjacent to lysine and arginine has been altered by replacing a glycine residue in the enzyme active site with an alanine, which carries an additional methyl group. This replacement favors the binding of lysine over the somewhat bulkier arginine. 

Fig. 15.47) (211). Subsequent SAR work led to the potent inhibitor SCH 66701 (115)(i i = 1.7 nM)y which was crystallized within the enzyme active site (212). This series of compounds is completely non-peptidic and also lacks the free sulfhydryl or imidazole seen in the other inhibitors discussed here. This is a breakthrough that shows that potency can be achieved even without the "essential" cysteine or sulfhydryl mimic. 

Activahon cascades, 64, 530 Activation peptide, 532 Active site, enzyme, 43 Adenomas, 146, 882-884,907 epideiruology, 907-908 /olale and, 908... 

---