7-Chymotrypsin, active center 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... 

A free -OH group of the tyro.syl residue is necessary for the activity of pepsin. Both the -OH of serine and the imidazole portion of histidine appear to be necessary parts of the active center of certain hydrolytic cn/ymes, such as trypsin ami chymotrypsin. and furnish the electrostatic forces involved in a proposed mechanism (Fig. 2S-3). in which E denotes enzyme and the other symbols are self-evident. (Alternative mechanisms have been propo.sed esterification and hydrolysis were studied extensively hy M. L. Bender sce Journal of the American Chemical Soeieiv 79 1258. IM7 80 5.3.38. 1958 82 1900. 1960 86 .3704. 53.30. 1964]. D. M. Blow reviewed studies concerning the structure and mechanism of chymotrypsin (.sec Accounts of Chemical Re-,twr<7i 9 145. 1976].)... 

These assumptions are confirmed by experiments with the spin-labeled active center of a-chymotrypsin [41] (Fig. 6). As seen from the data in Fig. 6, the enzyme entrapped in reverse micelles is located in the medium with decreased polarity. In similar polar media of water-organic mixtures, enzyme structures are normally disrupted, and protein denatura-tion and loss of catalytic enzymic activity occur. Yet, in systems of reverse micelles a principally different picture is observed. In optimal enzyme activity conditions the protein becomes tightly fixed by the micellar matrix and its conformational mobility is minimized. 

A second elastolytic enzyme (Af, 21,900) has been isolated from porcine pancreas. It shows higher activity than chymotrypsin in the hydrolysis of acetyltyrosine ester, which is used routinely to assay chymotrypsin. Another E,-like enzyme, a-lytic proteinase (Af, 19,900, 198 amino acids) has been isolated from the soil bacterium Myxobacter 495. This enzyme is remarkably similar to pancreatic E. both in structure (41 % homology, sequence in the active center Gly-Asp-Ser-Gly, 3 homologous disulfide bridges) and substrate specificity. Another E. (Af, 22,300) has been isolated from Pseudomonas aeruginosa. 

This biphenyl model compound was shown to possess the so-called primary optical specificity of a-chymotrypsin. That is, only the enantiomer related to L-phenylalanine was hydrolyzed by the enzyme. Generally speaking, there are three more levels of specific substrate recognition in enzyme catalysis. Let us consider a peptide bond in a polypeptide chain. The lateral side chain R2 is responsible for the normal specificity of the enzyme. For a-chymotrypsin, R2 is an aromatic side chain and the hydrophobic cavity a aromatic hole in the active center is there to accommodate the amino acid to be recognized by the enzyme. This is referred to as the primary structural specificity. 

Mild digestion of AMV RTase by chymotrypsin generates a 24-kDa polypeptide that exhibits only RNase H activity (77). Only the RNase H activity of the a subunit, but not that of a/8 complex, can be inhibited by captan, suggesting that there is some conformational change around the active center on a/3-complex formation (83). If the subunit structure of AMV RTase is considered similar to that of HlV-1 RTase (p66/p51 heterodimer), there is only one functional polymerase site within the asymmetric complex (96). 

Figure 14 Distance-geometry-generated structures of phenyl hippurate substrate bound to active site of chymotrypsin, using constraints showed in Figure 13 and described in text. For this and the stereo triples in Figures 16—19, the left and center views are for cross-eyed viewing, while the center and right are for a stereo viewer or wall-eyed (relaxed) viewing.

Although not macrocyclic structures, small peptides, not enzymes, used to catalyze organic reactions, should be briefly mentioned in the context of this chapter. Interest has centered for the greater part on hydrolytic reactions related to those catalyzed by, for example, chymotrypsin. Such studies usually involve the use of activated esters, usually p-nitrophenolates (7), which hydrolyze at readily followable rates under weakly basic conditions or in the presence of various nucleophiles. 

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