Other Hydrolytic Enzymes

Among the best known hydrolytic enzymes of which enough structural and mechanistic information are available are the exopeptidase carboxypeptidase A (see Chapter 6), ribonuclease A (see Chapter 3), and lysozyme. In this chapter we shall examine the chemistry of this last enzyme. 

Lysozyme is an important enzyme that catalyzes the hydrolysis of a polysaccharide that is the major constituent of the cell wall of certain bacteria. The polymer is formed from j8(l 4) linked alternating units of iV-acetyl-glucosamine (NAG) and iV-acetylmuramic acid (NAM) (Fig. 4.5). 

The enzyme is small, having a polypeptide chain of 129 amino acids. It was the first one, in 1967, to have its tertiary structure elucidated by X-ray crystallography (108). Unlike a-chymotrypsin, lysozyme has a well-defined deep cleft running down one side of the ellipsoidal molecule for binding the substrate. 

The cleft is divided into six subsites, ABCDEF. NAM residues can bind only in sites B, D, and F, while NAG residues of synthetic substrates may bind in all sites. The bond that is cleaved lies between sites D and E. 

The carboxyl group of Glu-35 in the unionized form and the carboxyl group of Asp-52 in its ionized form are the two functions implicated at the active site. 

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This chapter covers some general aspects of the use of enzymes in aqueous and organic media. Although lipases are the most common biocatalysts in these processes [4], other hydrolytic enzymes such as esterases and nitrilases have also shown their utility in the manufacture of pharmaceuticals. In addition, some representative examples using oxidoreductases and lyases will be also discussed. 

Although the hydrolysis of esters with lipases and esterases represents the most common process to obtain chiral intermediates for the synthesis of pharmaceuticals, proteases and other hydrolytic enzymes such as epoxide hydrolases and nitrilases have also been used for this purpose. We show here a few representative examples of the action of these biocatalysts that have been recently published. 

Two broad areas of application for xylanolytic enzymes have been identified (1). The first involves the use of xylanases with other hydrolytic enzymes in the bioconversion of wastes such as those from the forest and agricultural industries, and in the clarification and liquification of juices, vegetables and fruits. For these purposes, the enzyme preparations need only to be filtered and concentrated as essentially no further purification is required. Several specific examples of applications involving crude xylanase preparations include bioconversion of cellulosic materials for subsequent fermentation (2) hydrolysis of pulp waste liquors and wood extractives to monomeric sugars for subsequent production of single cell protein (3-5). Xylose produced by the action of xylanases can be used for subsequent production of higher value compounds such as ethanol (6), xylulose (7) and xyIonic acid (8-9). 

Although inversion was not observed with the E. colt alkaline phosphatase, it has been observed for ribonucleases and many other hydrolytic enzymes and for most kinases transferring phospho groups from ATP. The difference lies in the existence of a phospho-enzyme intermediate in the action of alkaline phosphatase (see Eq. 12-38). Each of the two phosphotransferase steps in the phosphatase action apparently occurs with inversion. The simplest interpretation of all the experimental results is that phosphotransferases usually act by in-line -like mechanisms which may involve metaphosphate-ion-like transition states that are constrained to react with an incoming nucleophile to give inversion. An adjacent attack with pseudorotation would probably retain the original configuration and is therefore excluded. 

There are other hydrolytic enzymes, such as lipases (see below) and alkaline phosphatase, with a mechanism closely related to that of the serine proteases or glyceraldehydephosphate dehydrogenase (GAP-DH) containing a cysteine in the active site. 

Lysosomes contain proteolytic and other hydrolytic enzymes. 

Sieben, A., Cellulase and other hydrolytic enzyme assays using an oscillating tube viscometer. Anal Biochem 1975, 63,(1), 214-9. 

Cellulases have been limited to a few specific applications, but economic and ecological factors have increased interest in their potential value. They have been used mainly as a component in digestive aids with other hydrolytic enzymes (Table VII). Toyama reported (62) in 1968 that commercial cellulase preparations were exported from Japan at a rate of 500 kg per month. 

Some other hydrolytic enzymes, in addition to proteases, that are important drug targets include protein phophatases, phosphodiesterases, nucleoside hydrolases, acetylhydolases, glycosylases, and phospholipases. Structure-based inhibitor design is currently being applied to a number of these enzymes. The last three mentioned have been successfully tar-... 

Catalytic Triads Are Found in Other Hydrolytic Enzymes... 

It appears that cobalt plays a particularly important role in the growth of cyanobacteria (Saito et al, 2002 Sunda and Huntsman, 1995b). Both Prochlorococcus and Synechococcus show an absolute cobalt requirement that zinc cannot substitute for (Figure 18(a)). The growth rate of Synechococcus is little affected by low zinc concentrations, except in the presence of cadmium which then becomes extremely toxic (Saito et al, personal communication). The biochemical processes responsible for the major cellular utilization of zinc and cobalt in marine cyanobacteria are unknown, however. These metals may be involved in carbonic anhydrase and/or other hydrolytic enzymes. Cobalamin (vitamin B12) synthesis is a function of cobalt in these organisms, yet B12 quotas tend to be very small (on the order of only 0.01 p.mol (mol C) ) and hence are not likely represent a significant portion of the cellular cobalt (Wilhelm and Trick, 1995). 

Liver acid phosphatase has been of particular interest since the demonstration by de Duve (D7, D8, D9, DIO) that acid phosphatase and other hydrolytic enzymes were enclosed in an intracellular structure, the lysosome, of the liver and played an important role in the intra-... 

The acid phosphatase appeared to be associated in the saclike structure with other hydrolytic enzymes, such as 3-glucuronidase and cathepsin, which also acted optimally at acid pH levels. Further studies were undertaken to isolate this structure (A13, G2). By means of a differential centrifugation procedure which will be described in detail later, de Duve and his associates (D9, DIO) determined the intracellular distribution of total and free acid phosphatase activity and of other enzymes as well. The mean values, expressed as percent of total acid phosphatase activity, were nuclear, 3.6 mitochondrial, 24.1 light mitochondrial, 40.7 microsomal, 20.1 final supernatant, 13.3. 

Phospholipase A2 (PLA2) hydrolyses phospholipids of cell membranes to release fatty acids such as arachidonic acid and thereby initiates the production of a diverse range of potent cellular mediators such as the prostaglandins (Slotboom et al., 1982). Verheij et al. (1980) proposed a mechanism which resembles the mechanism of other hydrolytic enzymes, in particular the serine proteases. According to this proposed mechanism (Figure 10., Verheij et al., 1980) a histidine (His-48 ofbovine pancreatic PLA2) and a... 

Bones are constantly subjected to forces that cause microscopic cracks. These microcracks (1) attach blood monocytes circulating within the periosteum and bone marrow and (2) induce adjacent osteoblasts to produce cytokines (Sect. 3.3.2) that cause these monocytes to proliferate, fuse, and differentiate into large multinucleated cells called osteoclasts. Osteoclasts cause bone resorption by acid demineralization and digestion of its proteins by enzymes that are optimally active in an acidic environment. These proteases and other hydrolytic enzymes are stored in a specialized, membrane-sealed compartment (lysosomes) into which they are guided by possessing terminal mannose 6-phosphate residues on N-linked glycans. 

The discovery of appropriate starting points for the development of potent and specific inhibitors is stiU a challenge. In addition to the usual problems of low abundance and purity that enzymologists and structural biologists are generally faced wifh, lipases present a unique additional difficulty. Unlike other hydrolytic enzymes, e.g. esterases or proteases, the substrates hydrolyzed by lipases are insoluble in water and therefore must be efficiently presented to the enzyme in a separate lipidic phase. The presence of a suitable second phase apparently leads to increased lipase activity and may effect subtle but critical changes to the enzyme s three-dimensional structure. 

Several systems have been identified in cells to mediate protein turnover through degradation. Lysosomes contain a number of proteases and other hydrolytic enzymes (Fig. 27.5). All of these enzymes are acid hydrolases and have an optimum activity at -pH 5. A primary role of lysosomes is to degrade proteins and other macromolecules that have been imported into the cell by endocytosis. Endocytosis is the process by which receptors on the cell surface... 

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