O

Figure 27.3 • Proposed generalized mechanism for enzyme-catalyzed hydrolysis of R—C—X.

Figure 27.4 • Generalized mechanism of protease catalysis. (Roberts, J. D.: Chem. Eng. News 57:23, 1979.)

The intermediate acylated enzyme is written with the proton on the imidazole nitrogen. The deacylation reaction involves the loss of this positive charge simultaneously with the attack of the nucleophilic reagent (abbreviated Nu:H).

Roberts20 used nitrogen-15 (15N) NMR to study the mechanism of protease catalysis. A schematic summary of the generalized mechanism is represented in Figure 27.4. It is concluded that the tertiary N-1 nitrogen of the histidine unit within the reactive center of the enzyme deprotonates the hydroxyl of the neighboring serine unit and simultaneously the hydroxyl oxygen exerts a nucleophilic attack on the carbonyl carbon of the amide substrate, as depicted in the scheme. A tetrahedral intermediate is implicated, and the carboxylate group of the aspartate unit (the third functional group within the reactive center) stabilizes the developing imidazolium ion by hydrogen bonding to the N-3 hydrogen. Finally, decomposition of the anionic tetrahedral intermediate toward product formation (amine and acylated serine) is promoted by prior protonation of the amide nitrogen by the imidazolium group.

A possible alternative route to deacylation would involve the nucleophilic attack of the imidazole nitrogen on the newly formed ester linkage of the postulated acyl intermediate, leading to the formation of the acyl imidazole. The latter is unstable in water, hydrolyzing rapidly to give the product and regenerated active enzyme.

The reaction of an alkyl phosphate in such a scheme may be written in an entirely analogous fashion, except that the resulting phosphorylated enzyme would be less susceptible to deacylation through nucleophilic attack. The diagrammatic scheme in Figure 27.5 has been proposed to explain the function of the active thiol ester site of papain. This ester site is formed and maintained by the folding energy of the enzyme (protein) molecule.

Zymogens (Proenzymes)

Zymogens, also called proenzymes, are enzyme precursors. These proenzymes are said to be activated when they are transformed to the enzyme. Activation usually involves catalytic action by some proteolytic enzyme. Occasionally, the activators merely effect a reorganization of the tertiary structure (conformation) of the protein so that the groups involved within the reactive center become functional (i.e., unmasked).

Synthesis and Secretion of Enzymes

Exportable proteins (enzymes), such as amylase, ribonucle-ase, chymotrypsin(ogen), trypsin(ogen), and insulin, are synthesized on the ribosomes. They pass across the membrane of the endoplasmic reticulum into the cisternae and directly into a smooth vesicular structure, which effects further transportation. They are finally stored in highly concentrated form within membrane-bound granules called zymogen granules. The exportable protein content of zymogen granules may reach a value of 40% of the total protein of the gland cell. In these enzyme sequences, the newly synthesized exportable protein (enzyme) is not free in the cell sap. The stored exportable digestive enzymes are released into the extracellular milieu and the hormones into adjacent capillaries. Release of these proteins is initiated by specific inducers. For example, cholinergic agents (but not epinephrine) and Ca2+ effect a discharge of amylase, li-pase, or others into the medium, increased glucose levels stimulate the secretion of insulin, and so on. This release of the reserve enzymes and hormones is completely independent of the synthetic process, as long as the stores in the granules are not depleted. Energy oxidative phosphorylation does not play an important role in these releases. Electron microscope studies indicate a fusion of the zymo-gen granule membrane with the cell membrane so that the granule opens directly into the extracellular lumen of the gland.

Classification

There are various systems for the classification of enzymes. The International Union of Biochemistry system includes some of the terminology used in the literature of

Figure 27.5 • Proposed scheme for the action of papain.

Figure 27.5 • Proposed scheme for the action of papain.

Papain Chemistry

medicinal chemistry, and in many instances the terms are self-explanatory. For example, transferases catalyze transfer of a group (e.g., methyltransferase); hydrolases catalyze hydrolysis reactions (e.g., esterases and amidases); and lyases catalyze nonhydrolytic removal of groups, leaving double bonds. There are also oxidoreductases, isomerases, and ligases. Other systems are sometimes used to classify and characterize enzymes, and the following terms are frequently encountered: lipase, peptidase, protease, phosphatase, kinase, synthetase, dehydrogenase, oxidase, and reductase.

Products

Pharmaceutically important enzyme products are listed in Table 27.3.

Pancreatin, USP. Pancreatin (Panteric) is a substance obtained from the fresh pancreas of the hog or the ox and contains a mixture of enzymes, principally pancreatic amy-lase (amylopsin), protease, and pancreatic lipase (steapsin). It converts not less than 25 times its weight of USP Potato Starch Reference Standard into soluble carbohydrates and not less than 25 times its weight of casein into proteoses. Pancreatin of higher digestive power may be brought to this standard by admixture with lactose, sucrose containing not more than 3.25% of starch, or pancreatin of lower digestive power. Pancreatin is a cream-colored amorphous powder with a faint, characteristic, but not offensive, odor. It dissolves slowly but incompletely in water and is insoluble in alcohol. It acts best in neutral or faintly alkaline media, and excessive acid or alkali renders it inert. Pancreatin can be prepared by extracting the fresh gland with 25% alcohol or with water and subsequently precipitating with alcohol. Besides the enzymes mentioned, it contains some trypsino-gen, which can be activated by intestinal enterokinase; chymotrypsinogen, which is converted by trypsin to chy-motrypsin; and carboxypeptidase.

Pancreatin is used largely for predigestion of food and for the preparation of hydrolysates. The value of its enzymes orally must be very small because they are digested by pepsin and acid in the stomach, although some of them may escape into the intestines without change. Even if they are protected by enteric coatings, it is doubtful they could be of great assistance in digestion.

Trypsin Crystallized, USP. Trypsin crystallized is a pro-teolytic enzyme crystallized from an extract of the pancreas gland of the ox, Bos taurus. It occurs as a white to yellowish

TABLE 27.3 Pharmaceutical^ Important Enzyme Products

Name

Proprietary Usual Adult Usual Dose

Name Preparations Category Application Dosea Rangea

TABLE 27.3 Pharmaceutical^ Important Enzyme Products

Name

Proprietary Usual Adult Usual Dose

Name Preparations Category Application Dosea Rangea

Pancreatin, USP

Pancreatin

Digestive aid

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