Chymotrypsin enzyme-substrate complex

Crystallographic studies (Blow, 1976) of the structure of the enzyme, enzyme-substrate complexes and enzyme-product complexes have identified a common feature in catalysis by the serine protease enzymes such as a-chymotrypsin. This is the well-known charge-relay system (44), in which... 

Chymotrypsin catalysis takes place through a three-step process, equation (11), where ES is an enzyme substrate complex which breaks down to give an acylated enzyme intermediate, ES and Pj,... 

The mechanism of action of chymotrypsin can be rationalized as follows (Figure 13.5). The enzyme-substrate complex forms, with the substrate being positioned correctly through hydrogen bonding and interaction with the pocket as described above. The nucleophilicity of a serine residue is only modest, but here it is improved by... 

Figure 3-2. Reaction mechanism of chymotrypsin as an example of covalent catalysis. Step I involves attack of the enzyme s active site serine on the peptide bond to be cleaved. In step II, a covalent complex is formed between the enzyme and a portion of the substrate (peptide 2) with release of the rest of the substrate (peptide I). Step III involves hydrolysis of the enzyme-substrate complex, which releases peptide 2 and completes the reaction.

Nature has provided a rare opportunity for determining the structures of the enzyme-substrate complexes of trypsin and chymotrypsin with polypeptides. 

When an ester such as acetyl-L-phenylalanine ethyl ester is mixed with a solution of chymotrypsin and proflavin, the following events occur. There is a rapid displacement of some of the proflavin from the active site as the substrate combines with the enzyme, leading to a decrease in A465. (This is complete in the dead time of the apparatus.) Then, as the acylenzyme is formed, the binding equilibrium between the ester and the dye is displaced, leading to the displacement of all the proflavin. The absorbance remains constant until the ester is depleted and the acylenzyme disappears. The dissociation constant of the enzyme-substrate complex may be calculated from the magnitude of the initial rapid displacement, whereas the rate constant for acylation may be obtained from the exponential second phase. 

The overall reaction mechanism of chymotrypsin is sketched in figure 8.11. Part a shows the enzyme-substrate complex, with an aromatic side chain of the substrate seated in the binding pocket and the carbonyl oxygen atom hy-... 

The probable mechanism of action of chymotrypsin. The six panels show the initial enzyme-substrate complex (a), the first tetrahedral (oxyanion) intermediate (b), the acyl-enzyme (ester) intermediate with the amine product departing (c), the same acyl-enzyme intermediate with water entering (d), the second tetrahedral (oxyanion)... 

Using this approach, Bizzozero and Zweifel (9) and Bizzozero and Dutler (10) have constructed molecular models of two intermediates (an enzyme-substrate complex and a tetrahedral intermediate) by appropriate modification of the models of stable enzyme-species. The stable enzyme-species used (15, 16) are trypsin-benzamidine complex (TR-B) (17), trypsin-pancreatic trypsin inhibitor complex (TR-PTI) (18, 19) and tosyl-chymotrypsin (Tos-CHT) (20) which are related to enzyme substrate complex, tetrahedral intermediate and acyl-enzyme respectively. 

Experimental support for the above mechanistic interpretation comes from the work of Bizzozero and Zweifel (9) who have studied the behavior of N-acetyl-j -phenyl al anyl- -prol i ne amide ( ) and N-acetyl-L-phenylalanyl-sarcosine amide (32) toward enzymic hydrolysis with o-chymotrypsin. These two dipeptides were found to be good competitive inhibitors with a specific substrate (Ac-Phe-0CH3 (33)) but no hydrolysis was observed. These two peptides thus form an enzyme-substrate complex and the reason for their nonreactivity has to be sought in the nature of the enzyme-substrate interactions occurring during the subsequent bond-change steps. 

Most of the enzymes show extremely strict chiral recognitions, and only one of the enantiomers can be the substrate of the enzyme. For example, chymotrypsin incorporates L-peptides only to the enzyme-substrate binding site to form enzyme-substrate complex, so it shows very high enantioselectivity (Figure 3 (a)). Oxidoreductases also form the enzyme-substrate complex of only one enantiomer, so enantioselectivities are high when isolated enzymes are used for reactions instead of whole cells containing both (R)- and (.S )-specific enzymes, which leads to overall low enantioselectivities. 

Blevins and Tulinsky (1985) suggested two functions for the solvent at the chymotrypsin active site (1) solvation of the Asp—His—Ser catalytic triad, and (2) a guiding effect on the substrate in formation of the enzyme-substrate complex, provided by several waters at the end of the specificity site. X-Ray diffraction results have suggested a role of active-site water in determining the kinetics or equilibria of substrate binding for other proteins (Section IV). 

Chymotrypsin catalyzes the hydrolysis of peptide bonds adjacent to aromatic amino acids. The probable mechanism for this reaction is illustrated in Figure 6.19. Step (a) of the figure shows the initial enzyme-substrate complex. Asp 102, His 57, and Ser 195 are aligned. In addition, the aromatic ring of the substrate s phenylalanine residue is seated in a hydrophobic binding pocket, and... 

At Stanford, Harden M. McConnell developed a new technique, called spin labelling, based upon EPR spectroscopy. While carbon-centered free radicals are extremely reactive and short-lived, radical oxides of nitrogen, such as NO and NO2, are moderately stable. McConnell noted that nitroxyl radicals (RR N-O) are extremely stable if R and R are tertiary and can be chemically attached to biological molecules of interest. In 1965, he published the concept of spin labeling and, in 1966, demonstrated that a spin-labelled substrate added to a-chymotrypsin forms a covalent enzyme-substrate complex. The EPR signal was quite broad suggesting restricted motion consistent with Koshland s induced-fit model. In 1971, McConnell published a smdy in which spin labelling indicated flip-flop motions of lipids in cell membranes. This was the start of dynamic smdies of cell membranes. 

With these introductory remarks, we may now consider the low-energy structures of enzyme-substrate complexes. In this section, we describe results for a-chymotrypsin and, in the next section, results for lysozyme. Further details may be found in a paper by Scheraga et al. [44]. 

All of these calculations are based on the use of ECEPP potentials [1,4], which have been obtained from crystal and gas-phase data. These potentials are ideally suited for computation of the structures of enzyme-substrate complexes because the interactions between enzymes and substrates are the same as those between the molecules of a crystal. They may therefore be employed (as we have done in the case of chymotrypsin and lysozyme) to identify the crucial interactions that lead to recognition. Once these interactions are known they may be used to construct, from theoretical considerations alone, substrates and inhibitors that can bind with the highest affinities to the active site of the enzyme. 

One of the closest approaches so far developed is by Bizzozero and Zweifel (118) who tried to explain in 1975 why a proline residue involved in a peptide bond is resistant to a-chymotrypsin cleavage. The objective was to find if the unreactivity of the peptide bond results from an unfavorable interaction of the methylene groups of the proline ring with the enzyme active site or whether the steric hindrance occurs upon formation of the enzyme-substrate complex or during the subsequent bond-change steps, and whether this steric hindrance is related to the ring structure of proline or simply to substitution of the amido nitrogen. In order to answer these questions, the dipeptides N-acetyl-L-phenylalanyl-L-proline amide and iV-acetyl-L-phenyl-alanyl-sarcosine amide were synthesized and their behavior as model substrates of a-chymotrypsin studied. 

In this latter case the Michaelis constant can be considerably smaller than the dissociation constant for the enzyme substrate complex, k2jkx2- The Michaelis constant defines the substrate concentration when half the enzyme, in the steady state, is in the form preceding the rate limiting step. Of course the situation can be less well defined when there are several steps with similar rates These comments are intended to underline the warning that Michaelis constants should not be used as measures for substrate affinities. A good example is the comparison of the values for the chymotrypsin catalysed hydrolysis of acetyl-L-tyrosine ethyl ester and the analogous amide. A hundredfold decrease in 23 results in an equivalent increase in... 

Chymotrypsin is the most-studied member of the serine protease family of enzymes. The enzyme-catalysed hydrolytic reaction has been shown to occur in at least three kinetically distinguishable steps. The first of these consists of a very fast, diffusion-controlled formation of a non-covalent enzyme-substrate complex, followed by an acylation step. In the latter the acyl group of the substrate is covalently attached to a serine alcohol of the active site with the concomitant release of the amine of an amide substrate, or the alcohol of an ester substrate. In a final deacylation step the acyl-enzyme intermediate is hydrolysed by water, thus regenerating the free enzyme and releasing the carboxylic acid ... 

Sometimes the mechanism of enzyme catalysis involves more than one enzyme-substrate complex. A representative example is chymotrypsin, one of the most-studied enzymes. Chymotrypsin can act as an esterase and a protease, because the chemical mechanisms of ester and amide hydrolases are almost identical. The catalytic mechanism when chymotrypsin acts as a serine protease involves the following steps ... 

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