Catalysis papain

An alternative to the synthesis of proteins by classical fragment synthesis in solution or by solid-phase synthesis on a support is the use of enzyme-catalyzed condensation of amino acids or peptides. This possibility was first demonstrated in 1938 91 with the synthesis of poorly soluble benzoyl-leucyl-leucine anilide by papain catalysis. After many years, this approach was extended to the preparation of peptide hormones such as Leu-enkephalin 92 and dynorphin(l -8).[93 This was made possible by the use of highly purified enzymes and by careful control of reaction conditions. The basic principles of protease-catalyzed peptide bond formation have been discussed.194 ... 

Figure 14, Analogy of formation of thiol adduct with aldehyde inhibitors to formation of tetrahedral intermediate in papain catalysis...

Wang et al. (1994) analyzed by MD the roles of the "double catalytic triad" in papain catalysis, based on the structure of the enzyme, which is not completely known from crystallography (Kamphuis et al., 1984) due to the oxidation state of Cys-25 (present as cysteic acid in the crystal). Stochastic boundary MD (Brooks and Karplus, 1983) was carried out on the whole enzyme + 350 water molecules. Three "layers" were treated according to their distance from the sulfur atom of Cys-25 - atoms within 12A, atoms between 12-16A and the more distant atoms were kept fixed. CHARMM forcefield was employed. The active site geometry was examined as a function of pH, for various mutual states of S-/SH and Im/ImH+. In addition, the mutations of Asp-158 (Menard et al., 1991) were studied. 

K. J. Angelides and A. L. Fink. Mechanism of thiol protease catalysis detection and stabilization of a tetrahedral intermediate in papain catalysis. Biochemistry 18 2663 (1979). 

Page, M. I., and Jencks, W. P., entropic hypothesis of enzyme catalysis, 224-225 Papain, Cys-His proton transfer in, 140-143 Pauling, Linus, view of enzyme catalysis, 208 PDLD model, see Protein dipoles-Langevin dipoles model (PDLD)... 

Reactive trajectories, 43-44,45, 88,90-92,215 downhill trajectories, 90,91 velocity of, 90 Relaxation processes, 122 Relaxation times, 122 Reorganization energy, 92,227 Resonance integral, 10 Resonance structures, 58,143 for amide hydrolysis, 174,175 covalent bonding arrangement for, 84 for Cys-His proton transfer in papain, 141 for general acid catalysis, 160,161 for phosphodiester hydrolysis, 191-195,... 

A family of 100 hybridoma antibodies can typically provide 20 tight binders and these need to be assayed for catalysis. At this stage in the production of an abzyme, the benefit of a sensitive, direct screen for product formation comes into its own. Following identification of a successful catalyst, the antibody is usually recloned to ensure purity and stabilization of the clone, then protein is produced in larger amount (—10 mg) and used for determination of the kinetics and mechanism of the catalysed process by classical biochemistry. Digestion of such protein with trypsin or papain provides fragment antibodies, Fabs, that contain only the attenuated upper limbs of the intact IgG (Fig. 1). It is these components that have been crystallized, in some... 

S. D. Lewis, F. A. Johnson, J. A. Shafer, Effect of Cysteine-25 on the Ionization of His-tidine-159 in Papain as Determined by Proton Nuclear Magnetic Resonance Spectroscopy. Evidence for a Hisl59-Cys25 Ion Pair and Its Possible Role in Catalysis , Biochemistry 1981, 20, 48-51. 

In enzymes, the most common nucleophilic groups that are functional in catalysis are the serine hydroxyl—which occurs in the serine proteases, cholinesterases, esterases, lipases, and alkaline phosphatases—and the cysteine thiol—which occurs in the thiol proteases (papain, ficin, and bromelain), in glyceraldehyde 3-phosphate dehydrogenase, etc. The imidazole of histidine usually functions as an acid-base catalyst and enhances the nucleophilicity of hydroxyl and thiol groups, but it sometimes acts as a nucleophile with the phos-phoryl group in phosphate transfer (Table 2.5). 

This text is a good source of information on the chemical mechanisms underlying the different modes of peptidase catalysis. Three important enzymes are covered subtilisin, a serine endopepti-dase papain, a cysteine endopeptidase and chymosin, an aspartic endopeptidase. 

Cysteine proteases are a class of enzymes that have been widely studied over the years. The overall principles of substrate recognition, catalysis, and inhibition are now reasonably well documented. This enzyme class includes the plant proteases such as papain, actinidin, and bromelain, and several mammalian lysosomal cathe-psins. By far the majority of the literature reports dealing with cysteine proteases describe results obtained with the enzyme papain, because it is considered to be the archetype of this enzyme class. 

Figure 5.8 Amide hydrolysis in the presence of papain, an example of nucleophilic enzyme catalysis.

Bromelain differs from (be other cysieinyl proteases papain and ficin in its 140-told difference of Itcat for the BAEK and BAA hydrolysis, suggesting a difference in the mechanism of catalysis for both substrates [37]. For BABE hydrolysis, deacylation is predominantly the rate-limiting step, while for BAA hydrolysis (he acylation is rate limiting [42]. However, Wharton et aL [43] explained the differences in kcot for BAEE and BAA hydrolysis a gauming (hat nonproductive binding plays a role in catalysis. 

The —CTanion in Ser plays the role of the catalysis so that a-chymotrypsin is a member of the class of enzymes called serine proteinase . We have many serine proteinase such as tripsin, elastase, subtilicin, etc. Papain is called thiol proteinase and contains thiol moiety in place of serine. 

One of the key intermediates shown in this reaction scheme is the formation of a tetrahedral adduct during acylation and deacylation (84). Additional support for the formation of a tetrahedral intermedite comes from the observation already referred to— that aldehydes may act as potent inhibitors of papain. Westerik and Wolfenden (65) attribute the inhibitory eflFect of aldehydes to the formation of a stable thiol adduct (thiohemiacetal) analogous to the tetrahedral intermediate produced when papain acts on a substrate. This relationship is depicted in Figure 14. When the complete picture for the mechanism of catalysis by the thiol proteases finally emerges, it will no doubt be similar to the mechanism of action of the serine proteinases. 

Panella L, Broos J, Jin JF, Fraaije MW, Janssen DB, Jeronimus-Stratingh M, Feringa BL, Minnaard AJ, de Vries JG. Merging homogeneous catalysis with biocatalysis papain as hydrogenation catalyst. Chem. Commun. 2005 45 5656-5658. 

The large size of the pores of MCM-41 has also allowed the entrapment of enzymes, such as cytochrome c, papain and trypsin [193]. Enzyme entrapment has been extensively performed with sol-gel materials. The types of applications of redox catalysis using enzyme-mesoporous materials is expected to parallel the sol-gel materials, which is discussed in the last section of this chapter. 

Papain is widely used in brewing industry, for tenderization in meat industry, fish, food, laundry, detergents, pharmaceutical and allied indushies. Papain consists of a single polypeptide chain of 212 amino acid residues, cross-linked by four disulphide bonds. The active site of papain contains a cysteine residue whose sulphhydryl group is required for catalysis. It has a broad specificity for peptide bonds with wide pH range. 

As an example, consider papain, a protease from papaya juice that is used to treat meat to make it more tender and easier to chew. The pH optimum of the enzyme is 6.2, and analysis of the pH dependence indicates that two groups, with pKg s of 4.2 and 8.2, are required for catalysis. One group must be protonated cmd the other deprotonated for catalysis to occur. The active site of papain contains a cysteine thiol and a histidine imidazole group. There are two possible ways to write the protonation state of the active site ... 

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