Chymotrypsin, enzymatic mechanisms

The ability of the quantum chemical approaches to analyze, interpret and rationalize all experimental observations on molecular processes in a unified language based on the primary principles of physics, represents an important asset in the study of biological systems where experimental data has to be obtained in a variety of forms and from many different sources. Taking advantage of this formal and anlytic power of the quantum chemical approaches, Lipscomb and his coworkers studied models of enzymatic mechanisms in chymotrypsin (21) and caboxypeptIdase (22). 

The catalytic efficiency of enzymes is a subject of great fascination, particularly when crystallographic structures are available and a great deal is known about the physical organic chemistry of the enzymatic mechanism. In this regard, the most studied enzyme of a group of enzymes called serine proteases is a-chymotrypsin. The term serine protease derives from the fact that this class of enzymes contain at their active site a serine hydroxyl group which exhibits unusual reactivity toward the irreversible inhibitor diiso-propylphosphorofluoridate (DFP). 

Azaserine, 5-diazo-4-oxo-L-norvaline (DONV) and 6-diazo-5-ketonorleucine (DON) are other examples of mechanism-based irreversible inhibitors51). They are stable to nucleophilic attack, but on enzymatic protonation, they are converted to the reactive diazonium ions (30). N-Nitroso compounds have been proposed as irreversible inhibitors of proteolytic enzymes. N-Nitrosolactam (31) can inhibit chymotrypsin... 

Measurements of the temperature dependence of enzymatic activity suggested the presence of an intermediate step in enzyme inactivation. The proposed mechanism involves a reversible change to an inactive state, which precedes the final irreversible inactivation step [21-23]. This mechanism could now be refined based on experiments with single enzymes, which detected the intermediate steps directly. This more detailed information allowed the establishment of a tentative model for a-chymotrypsin inactivation (Fig. 25.2d). 

Polyethylene glycol is used to make the enzymes soluble in organic solvents [88], and high reaction yields have been obtained with polyethylene-glycol-modified chymotrypsin [89], and papain in benzene [90], Enzymatic modification reactions with deacylation, via aminolysis, of an intermediate covalent acyl-enzyme also support the mechanism of transpeptidation in kinetically controlled peptide syn-... 

The inventory technique is clearly a potentially useful tool for elucidating enzymatic transition states. Deacetylation of acetyl-a-chymotrypsin is consistent with a single proton transfer (Eqn. 45) [25] with a fractionation factor 0.42. It is thought that this result excludes the charge-relay mechanism (Eqn. 46) which would require a quadratic term for the dependence. 

The mechanism of chymotrypsin action is particularly well studied and, in many respects, typical. Numerous types of reaction mechanisms for enzyme action are known, and we shall discuss them in the contexts of the reactions catalyzed by the enzymes in question. To lay the groundwork, it is useful to discuss some general types of catalytic mechanisms and how they affect the specificity of enzymatic reactions. 

The overall mechanism for a reaction may be fairly complex, as we have seen in the case of chymotrypsin, but the individual parts of a complex mechanism can themselves be fairly simple. Concepts such as nucleophilic attack and acid catalysis commonly enter into discussions of enzymatic reactions. We can draw quite a few general conclusions from these two general descriptions. 

Acyl enzyme, an intermediate in the catalytic mechanism of serine proteases, such as trypsin and chymotrypsin. After the serine protease has bound a peptide substrate to form the Michaelis complex, Ser (in the case of chymotrypsin) nucleophilically attacks the peptide bond in the rate-determining step, forming a transition-state complex, known as a tetrahedral intermediate. The latter decomposes to the acyl enzyme, an extremely unstable intermediate, that bears the acyl moiety at the hydroxy group of Ser . The acyl enzyme intermediate is deacylated by water during proteolysis, or the attacking nucleophile is an amino component in case of kineticaUy controlled enzymatic peptide synthesis. 

Chymotrypsin, trypsin, and elastin are digestive enzymes secreted by the pancreas into the small intestine to catalyze the hydrolysis of peptide bonds. These enzymes are all called serine proteases because the mechanism for their proteolytic activity (one that they have in common) involves a particular serine residue that is essential for their enzymatic activity. As another example of how enzymes work, we shall examine the mechanism of action of chymotrypsin. 

Enzyme reaction intermediates can be characterized, in sub-second timescale, using the so-called pulsed flow method [35]. It employs a direct on-line interface between a rapid-mixing device and a ESI-MS system. It circumvents chemical quenching. By way of this strategy, it was possible to detect the intermediate of a reaction catalyzed by 5-enolpyruvoyl-shikimate-3-phosphate synthase [35]. The time-resolved ESI-MS method was also implemented in measurements of pre-steady-state kinetics of an enzymatic reaction involving Bacillus circulans xylanase [36]. The pre-steady-state kinetic parameters for the formation of the covalent intermediate in the mutant xylanase were determined. The MS results were in agreement with those obtained by stopped-flow ultraviolet-visible spectroscopy. In a later work, hydrolysis of p-nitrophenyl acetate by chymotrypsin was used as a model system [27]. The chymotrypsin-catalyzed hydrolysis follows the mechanism [27] ... 

The reaction mechanisms of some enzymatic reactions are known in detail due to the development of powerful structure elucidation tools, such as X-ray and NMR enzymatic reaction mechanisms are no longer in a black box. One of the most studied hydrolytic enzymes is chymotrypsin, which represents a group of serine proteases. It catalyzes the hydrolysis of peptides to amino acids, and the reaction mechanism is shown in Fig. 10.3. Two amino acid residues of the enzyme. Asp and His, locate together to facilitate nucleophilic attack of Ser on the carbonyl carbon of the substrate. The reaction proceeds through a tetrahedral transition state, cleavage of the peptide bond and rapid diffusion of the amine moiety to leave the acyl-enzyme intermediate, foUowedby hydro lysis to give a carboxyhc acid. 

---