Chymotrypsin crystal

To understand how hexane may affect the protein, we analyzed the solvent interaction with the protein. Six of the seven initially "bound" hexane molecules in were found to have diffused off of the protein surface at the end of the dynamics run. The one bound hexane (Hex 238) remaining is surrounded by several large hydrophobic residues forming a stable hydrophobic pocket on the protein surface. Interestingly, this hexane site was again observed in a subsequent chymotrypsin crystal grown and then exposed to hexane and isopropanol mixtures by Farber and co-workers. This hexane site was not occupied in the CTWAT and CTMONO systems and no hexane molecules were found in other interior regions of the protein. 

Inhibitors as well as substrates bind in this crevice between the domains. From the numerous studies of different inhibitors bound to serine pro-teinases we have chosen as an illustration the binding of a small peptide inhibitor, Ac-Pro-Ala-Pro-Tyr-COOH to a bacterial chymotrypsin (Figure 11.9). The enzyme-peptide complex was formed by adding a large excess of the substrate Ac-Pro-Ala-Pro-Tyr-CO-NHz to crystals of the enzyme. The enzyme molecules within the crystals catalyze cleavage of the terminal amide group to produce the products Ac-Pro-Ala-Pro-Tyr-COOH and NHs. The ammonium ions diffuse away, but the peptide product remains bound as an inhibitor to the active site of the enzyme. 

Fujinaga, M., et al. Crystal and molecular structures of the complex of a-chymotrypsin with its inhibitor turkey ovomucoid third domain at 1.8 A resolution. 

Laufberger had tried to obtain the protein from horse liver, but it did not crystallize, and as he described to me when I met him in Prague some years ago, in those days everyone wanted to have protein crystals as a criteria of purity. Although James Sumner had crystallized jack bean urease in 1926, his preparations were somewhat impure, and it was only in the mid-1930s, when John Northrop and Moses Kubnitz showed that there is a direct correlation between the enzymatic activities of crystalline pepsin, trypsin and chymotrypsin that the protein nature of enzymes was generally accepted. 

Globular proteins were much more difficult to prepare in an ordered form. In 1934, Bernal and Crowfoot (Hodgkin) found, that crystals were better preserved if they were kept in contact with their mother liquor sealed in thin-walled glass capillaries. By the early 1940s crystal classes and unit cell dimensions had been determined for insulin, horse haemoglobin, RNAase, pepsin, and chymotrypsin. Complete resolution of the structures required identification of the crystal axes and some knowledge of the amino acid sequence of the protein—requirements which could not be met until the 1950s. 

Sigler, P. B. (1970). lodination of a single tyrosine in crystals of alpha-chymotrypsin. Biochem. 9,3609. 

The strongest influence of configuration has been observed for Pi -substituted diastereomers of Z-Phe-(aTfm)Ala-Ala-NH2. The crystal structures of both dia-stereomers have been solved, which enables a better interpretation of this rather interesting effect. While the (S,S,S)-diastereomer has been shown to be almost as stable as the Aib-substituted peptide, the (S,R,S)-diastereomer was hydrolyzed very quickly within the same time range. Molecular modeling studies readily support the formation of hydrogen bonds as a possible explanation for this effect [18,54]. With the known crystal structure of the a-chymotrypsin/phenyl boronic... 

In subsequent years, much evidence has been adduced to support this mechanism. Alkaline phosphatase and, by analogy, other serine enzymes, are directly phosphorylated on serine serine phosphate is not an artifact (Kennedy and Koshland, 1957). In the presence of nitrophenyl acetate, chymotrypsin is acetylated on serine, and the resulting acetylchymotrypsin has been isolated (Balls and Aldrich, 1955 Balls and Wood, 1956). Similarly, the action of p-nitrophenyl pivalate gave rise to pivaloyl chymotrypsin, which could be crystallized (Balls et al., 1957). Neurath and workers showed that acetylchymotrypsin is hydrolyzed at pH 5.5, but that it is reversibly denatured by 8 M urea the denatured derivative is inert to hydrolysis and even to hydroxylamine, whereas the renatured protein, obtained by... 

The mammalian serine proteases appear to represent a classic case of divergent evolution. All were presumably derived from a common ancestral serine protease.23 Proteins derived from a common ancestor are said to be homologous. Some nonmammalian serine proteases are 20 to 50% identical in sequence with their mammalian counterparts. The crystal structure of the elastase-like protease from Streptomyces griseus has two-thirds of the residues in a conformation similar to those in the mammalian enzymes, despite having only 186 amino acids in its sequence, compared with 245 in a-chymotrypsin. The bacterial enzymes and the pancreatic ones have probably evolved from a common precursor. 

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. 

The structural basis of one of the classic examples of such stereospecificity, that of chymotrypsin for L-amino acid derivatives, is immediately obvious on examination of the crystal structure of the enzyme. D-Amino acid derivatives differ from those of L-amino acids by having the H atom and the side chain attached to the chiral carbon interchanged (structure 8.10). The d derivatives cannot bind because of steric hindrance between the side chain and the walls of enzyme around the position normally occupied by the H atom of L derivatives (Chapter 1). 

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... 

But where there is an equilibrium among two or more conformations of the enzyme in solution, crystallization may select out only one of the conformations. a-Chymotrypsin has a substantial fraction of an inactive conformation present under the conditions of crystallization, but only the active form of the enzyme crystallizes. An allosteric effector molecule that changes the conformation of the protein in solution may have no effect on the crystalline protein, as, for example, with phosphorylase b.5A The enzyme is frozen in one conformation, with the crystal lattice forces preventing any conformational change. On the other hand, the addition of an effector to phosphorylase a causes the crystals first to crack and then to anneal, giving crystals of the enzyme in a second conformation. 

The crystal structures also provided an explanation for the different substrate specificities of trypsin, chymotrypsin,... 

Protein aggregates [107, 109, 121] or dye crystals [122-126] can serve as templates for LbL polyelectrolyte adsorption. Chymotrypsin aggregates encapsulated by PSS and PAH deposition contain a high protein amount and the enzyme keeps its bioactivity [107], The aggregates prepared in this manner have high incorporation efficiency and a protein content of 50-70% [109]. An encapsulated catalase has been shown to be stable against protease degradation [121],... 

The third approach to solving this problem (Farber, 1999) involves the preparation of an enzyme-intermediate complex at high substrate concentration for X-ray data collection. Under such a condition active sites in the crystal lattice will be filled with intermediates. Using a combination of flow cell experiments and equilibrium experiments, it is possible to obtain the structure of important intermediates in an enzyme reaction (Bolduc et al., 1995). It was also discovered that some enzyme crystals can be transformed from their aqueous crystallization buffer to nonaqueous solvents without cross-linking the crystals before the transfer (Yennawar et. al., 1995). It is then possible to regulate the water concentration in the active site. The structure of the first tetrahedral intermediate, tetrapeptide -Pro-Gly-Ala-Tyr- in the y-chymotrypsin active site obtained by this method is shown in Fig. 1.1. 

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