Chymotrypsin acylenzyme

For example, chymotrypsin reacts with /j-nitrophenyl acetate (AcONp) according to the above scheme (when [AcONp] < Ks for the first step) to give an intermediate acylenzyme, EAc ... 

The currently accepted mechanism for the hydrolysis of amides and esters catalyzed by the archetypal serine protease chymotrypsin involves the initial formation of a Michaelis complex followed by the acylation of Ser-195 to give an acylenzyme (Chapter 1) (equation 7.1). Much of the kinetic work with the enzyme has been directed toward detecting the acylenzyme. This work can be used to illustrate the available methods that are based on pre-steady state and steady state kinetics. The acylenzyme accumulates in the hydrolysis of activated or specific ester substrates (k2 > k3), so that the detection is relatively straightforward. Accumulation does not occur with the physiologically relevant peptides (k2 < k3), and detection is difficult. 

In 1954, B. S. Hartley and B. A. Kilby1 examined the reaction of substrate quantities of chymotrypsin with excess p-nitrophenyl acetate or p-riitrophenyl ethyl carbonate. They noted that the release of p-nitrophenol did not extrapolate back to zero but instead involved an initial burst, equal in magnitude to the concentration of the enzyme (Chapter 4, Figure 4.10). They postulated that initially the ester rapidly acylated the enzyme in a mole-to-mole ratio, and that the subsequent turnover of the substrate involved the relatively slow hydrolysis of the acylenzyme as the rate-determining step. This was later verified by the stopped-flow experiments described in section B2. 

Chromophoric acyl group,4,5 The spectrum of the furylacryloyl group depends on the polarity of the surrounding medium, and also on the nature of the moiety to which it is attached. The spectrum of furylacryloyl-L-tyrosine ethyl ester changes slightly when it is bound to chymotrypsin. There are also further changes on formation of the acylenzyme and on the subsequent hydrolysis. The rate constants for acylation and deacylation and the dissociation constant of the Michaelis complex may be measured by the appropriate experiments. 

There are also nonthematic methods that allow the formation of acylenzymes under conditions where they are stable, so that they can be stored in a syringe in a stopped-flow spectrophotometer. For example, it is possible to synthesize certain nonspecific acylenzymes and store them at low pH.9 12 When they are restored to high pH, they are found to deacylate at the rate expected from the steady state kinetics. This approach has been extended to cover specific acylenzymes. When acyl-L-tryptophan derivatives are incubated with chymotrypsin at pH 3 to 4, the acylenzyme accumulates. The solution may then be pH-jumped by mixing it with a concentrated high-pH buffer in the stopped-flow spectrophotometer.1314 The deacylation rate has been measured by the proflavin displacement method and by using furylacrylolyl compounds. 

The preceding experiments prove that there is an intermediate on the reaction pathway in each case, the measured rate constants for the formation and decay of the intermediate are at least as high as the value of kcat for the hydrolysis of the ester in the steady state. They do not, however, prove what the intermediate is. The evidence for covalent modification of Ser-195 of the enzyme stems from the early experiments on the irreversible inhibition of the enzyme by organo-phosphates such as diisopropyl fluorophosphate the inhibited protein was subjected to partial hydrolysis, and the peptide containing the phosphate ester was isolated and shown to be esterified on Ser-195.1516 The ultimate characterization of acylenzymes has come from x-ray diffraction studies of nonspecific acylenzymes at low pH, where they are stable (e.g., indolylacryloyl-chymotrypsin),17 and of specific acylenzymes at subzero temperatures and at low pH.18 When stable solutions of acylenzymes are restored to conditions under which they are unstable, they are found to react at the required rate. These experiments thus prove that the acylenzyme does occur on the reaction pathway. They do not rule out, however, the possibility that there are further intermediates. For example, they do not rule out an initial acylation on His-57 followed by rapid intramolecular transfer. Evidence concerning this and any other hypothetical intermediates must come from additional kinetic experiments and examination of the crystal structure of the enzyme. 

Figure 16 4 The crystal structure of indolylacryloyl-chymotrypsin. [From R. Henderson, J. Molec. Biol. 54, 341 (1970).] Note that the carbonyl oxygen of this nonspecific acylenzyme is not bound between the NH groups of Ser-195 and Gly-193, but is nonproductively linked to His-57 by a hydrogen-bounded water molecule. This is the acylenzyme that was found to deacylate at the same rate in solution and in the crystal (Chapter 1). [G. L. Rossi and S. A. Bernhard, J. Molec. Biol. 49, 85 (1970).]...

In many cases, the crystal retains enzymatic activity. In some cases, the activity of the enzyme in the crystal is the same as that in solution. The methods used for initiating reactions for study by the Laue method are used to measure activity. For example, pH-jump the acylenzyme indolylacryloyl-chymotrypsin was crystallized at a pH at which it is stable. On changing the pH to increase the reactivity, the intermediate was found to hydrolyze with the same first-order rate constant as occurs in solution the reactions of crystalline ras p21 protein, glycogen phosphorylase, and chymotrypsin have been initiated by photolysis.52 Glyceraldehyde 3-phosphate dehydrogenase has also identical reaction rates in the crystal and solution under some conditions.53... 

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 hydrolysis of esters (and amides) by chymotrypsin satisfies these criteria. The hydrolysis of, say, acetyl-L-tryptophan p-i itrophenyl ester forms an acylen-/yme that reacts with various amines such as hydroxylamine, alaninamide, hydrazine, etc., and also with alcohols such as methanol, to give the hydroxamic acid, dipeptide, hydrazide, and methyl ester, respectively, of acetyl-L-tryptophan. The same acylenzyme is generated in the hydrolysis of the phenyl, methyl, ethyl, etc., esters of the amino acid (and also during the hydrolysis of amides). 

Figurel.10. Stereo diagram of the acyl moiet of the spin-labeled tryptophanyl-acylenzyme reaction intermediate of a-chymotrypsin. Active site residues close to the acyl moiety are labeled (Makinen et al., 1998). Reproduced with permission.

These form the most studied class of peptidases. They have a reactive serine residue, e.g. the hydrolysis of a peptide substrate involves an acylenzyme intermediate in which the hydroxyl group of Ser195 (from the chymotrypsin numbering system) is acylated by the acyl moiety of the substrate, releasing the amine fragment of the substrate as the first product. The formation of the acylenzyme is the slow step in peptide bond hydrolysis, but the acylenzyme often accumulates in the hydrolysis of ester substrates. The acylenzyme thus formed will be the same for a series of substrates which differ in their leaving group. 

J.24 The reaction of either the E isomer (1) or the Z isomer (2) leads to the acylenzyme, probably a-benzamidocinnamoyl-a-chymotrypsins... 

A simplified reaction mechanism for chymotrypsin is shown in Figure 6.11. The carbonyl carbon atom of the peptide bond to be hydrolysed is subject to attack by the polarized oxygen atom of serine-195. An acylenzyme intermewater molecule then enters and is polarized by the charge relay system. The hydroxyl residue attacks the carbonyl carbon of the acyl group attached to serine-195, with the hydrolysis of the acylenzyme intermediate and release of the product. 

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