Hydrolytic catalytic activity enzymes

Table I. Hydrolytic catalytic activity, Kd (sec ), of selected enzymes toward some nerve agents...

Lysosomes are membrane-bound organelles that contain hydrolytic enzymes to break down macromolecules and other organelles taken up by the lysosomes. The pH within this organelle is very low (about 5.0) and the catalytic activities of the enzymes, within it, are highest at this pH. The pH in the cytosol is about 7.1, so that any enzymes released from the lysosome are not catalyticaUy active in the cytosol. 

By rect comparison of structural and functional characteristics of different, naturally-occurring enzymes and modified biocatalysts, researchers hope to delineate the structural features controlling the hydrolytic and S3mthetic catalytic activities of this family of glucosylases. This understanchng will eventually lead to the ability to "engineer" glucan biocatalyst capability. 

The secondary structure of the polypeptide chain in hydrolytic enzymes ensures the spatial proximity of the necessary functional groups, which are responsible for the observed catalytic effect. In synthetic enzyme mimics, it is possible to bring the requisite functionalities into close juxtaposition only if there is a rigid framework to which these groups are attached. It was thus logical to examine cyclic peptides, especially cyclodipeptides, which bear the necessary functional groups for their catalytic activity in ester hydrolysis. 

A host of enzymes, which are described elsewhere in the book, act on DNA and RNA. They include hydrolytic nucleases, methyltransferases, polymerases, topoisomerases, and enzymes involved in repair of damaged DNA and in modifications of either DNA or RNA. While most of these enzymes are apparently proteins, a surprising number are ribozymes, which consist of RNA or are RNA-protein complexes in which the RNA has catalytic activity. 

Ribonuclease-S can be separated into S-peptide [residues 1-20 (21)] and S-protein [residues 21 (22)-124] by precipitation with trichloroacetic acid 73) or better, Sephadex chromatography in 5% formic acid 83). The best preparations of these components show no detectable hydrolytic enzymic activity and little if any transphosphorylation activity (see Section VI). Isolated S-peptide appears to have no regular secondary structure 83, 84) or 10-20% helicity 85, 86). (These slightly different interpretations are based on almost identical CD data.) When equimolar amounts of S-protein and S-peptide are mixed at neutral pH and room temperature or below, essentially full catalytic activity is recovered 73, 87). A schematic diagram is shown in Fig. 7. For a detailed summary of the preparative procedures see Doscher 88). 

Molenveld P et al (1999) Dinuclear and trinuclear Zn(II) calix[4]arene complexes as models for hydrolytic metallo-enzymes. Synthesis and catalytic activity in phosphate diester transesterification. J Org Chem 64 3896-3906... 

FIGURE 4. Molecular model of the peptide backbone of silicatein a (221 amino acid residues, constrained by three intramolecular disulfide cross-links), determined as described in the text. Locations of the putative catalytically active serine (at position 26) and histidine (at position 165) in juxtaposition on both sides of the active-site (substrate binding) cleft are identified. These features are very similar to those in the homologous protease (hydrolytic enzyme)... 

True lipases show the interfacial activation phenomenon in their catalytic activity pattern. At low concentration of water-insoluble substrates, lipases are almost inactive, and the hydrolytic activity does not increase linearly. At a certain substrate concentration, however, the hydrolytic activity of lipases increases rapidly and the lipase kinetics resembles normal enzyme kinetics. This boost in activity is related to the formation of water-insoluble substrate aggregates such as micelles or another second phase. Only when this second phase is present, do lipases become fully active. This interfacial activation is caused by a large conformational change in the 3D structure of the lipases. In their water-soluble form, the active site is covered by a lid, which prevents the substrates from reaching it. At the lipidAvater interface, the lid is opened and the active site is accessible to the substrates. In addition, the now accessible area is mainly hydrophobic, which gives the open-form lipase the shape and behavior of conventional surfactant molecules with a hydrophilic and a hydrophobic moiety in one single molecule. 

Metal ions are vital to the function of many enzymes that catalyze hydrolytic reactions. Coordination of a water molecule to a metal ion alters its acid-base properties, usually making it easier to deprotonate, which can offer a ready means for catalyzing a hydrolytic reaction. Also, the placement of a metal center in the active site of a hydrolytic enzyme could permit efficient delivery of a catalytic water molecule to the hydrolyzable substrate. In fact, the first enzyme discovered, carbonic an-hydrase, is a metalloenzyme that requires a Zn2+ center for its catalytic activity (32). The function of carbonic anhydrase is to catalyze the hydrolysis of carbon dioxide to bicarbonate ... 

Two different types of enzymatic time-temperature integrators are described. The first, under the tradename of I-point, is based on a lipase-catalyzed hydrolysis reaction (125). The lipase is stored in a nonaqueous environment containing glycerol. The indicator contains two components that are mixed when the indicator is activated. The operating principle is as follows Upon activation, two volumes of reagents are mixed with each other. Lipase is thereby exposed to its substrate, here a triglyceride. At low temperatures there will be almost no hydrolytic reaction. As the temperature increases, hydrolysis accelerates and protons are liberated. A pH indicator is dissolved in the system. The indicator is selected to shift color after a certain amount of acid has been liberated by the enzyme-catalyzed process. Since the catalytic activity is influenced both by temperature and time, this indicator strip is said to be a time-temperature integrator. 

The charge relay system is found at the active site of a group of enzymes called serine proteases. They include chymotrypsin, trypsin, a-lytic protease, elastase, and subtilisin. It is interesting that the charge relay system was found in enzymes belonging to different branches of diemical evolution (chymotrypsin and subtilisin). This suggests that this system is a hydrolytic catalytic system of general importance which is derived solely from amino acid residues. 

Although polyethyleneimine possesses moderate catalytic effects on several hydrolysis reactions, the polymer modified with long allQrl chains show mudi enhanced reactivity toward hydrophobic substrates. Remaikable catalytic effects were observed in the hydrolysis of phenyl esters and a sulfate ester catalyzed by the methylene-imidazole-modified polymer. These results were explained as arising from the hi y brandied, compact stmcture of the polyediyleneimine derivative. Klotz called the imidazole-containing polyethyleneimine synzyme (synthetic enzyme), on the basis of its stmctural characteristics and catalytic activity which are comparable to hydrolytic enzymes. 

The carbapenems are mechanism-based inhibitors which involve acylation of the active-site residue and subsequent rearrangement to a more stable acyl-enzyme species. Knowles and co-workers [32, 33] have demonstrated that the progressive inhibition of the TEM S-lactamase by the olivanic acids is due to the rearrangement of the J -pyrroline intermediate (15) to the tautomeric and thermodynamically more stable zl -pyrroline (16) Scheme 6.3). The resultant acyl-enzyme complex is believed to be stable to subsequent hydrolytic breakdown, thereby disrupting the catalytic activity of the enzyme. 

Cross-linked enzyme crystals (CLEC, [63]) can have greatly improved catalytic activity over native enzyme. Their practical utility has recently been demonstrated for the hydrolytic kinetic resolution of the acetates of 1-phenylethanol (Figure 3.28) [64]. The catalyst was recovered and recycled eight times, on a 100-liter scale. The future will show increased use of enzymes. 

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