Models hydrolytic enzyme

A frequently employed strategy to generate more effective hydrolytic enzyme models is the use of bi- or even trinuclear metal complexes. The active center of natural enzymes often contains two or three divalent metal ions, lying close together with a metal-metal distance of a variety of biomimetic bi- or trinuclear... 

Catalysis by imidazole in aqueous systems has received widespread attention because of its central position as the catalytic group in many hydrolytic enzymes. Many imidazole derivatives with long aliphatic chains have been synthesized and their catalytic role in the presence of detergents has been reported as models of hydrolytic enzymes. Representative examples of the hydrolysis ofp-nitrophenyl acetate (8) are summarized in Table 2. 

The three hydrolytic enzymes that have been discussed, a-chymo-trypsin, carboxypeptidase A, and lysozyme, cover a wide range of substrate types and mechanistic possibilities. Formulation of principles which might apply to enzymatic catalysis in general is difficult from such a small sampling, but certain features of the enzymatic and model reactions warrant some comment. 

In the area of catalysis, the esterolysis reactions of imidazole-containing polymers have been investigated in detail as possible models for histidine-containing hydrolytic enzymes such as a-chymotrypsin (77MI11104). Accelerations are observed in the rate of hydrolysis of esters such as 4-nitrophenyl acetate catalyzed by poly(4(5)-vinylimidazole) when compared with that found in the presence of imidazole itself. These results have been explained in terms of a cooperative or bifunctional interaction between neighboring imidazole functions (Scheme 19), although hydrophobic and electrostatic interactions may also contribute to the rate enhancements. Recently these interpretations, particularly that depicted in Scheme 19, have been seriously questioned (see Section 1.11.4.2.2). 

In the preceding section we discussed the use of co-ordinated hydroxide as an intramolecular nucleophile. It could also act as a nucleophile to an external electrophile. Over the past few decades, there has been considerable interest in the nucleophilic properties of metal-bound hydroxide ligands. One of the principal reasons for this relates to the widespread occurrence of Lewis acidic metals at the active site of hydrolytic enzymes. There has been a lively discussion over the past thirty years on the relative merits of mechanisms involving nucleophilic attack by metal-co-ordinated hydroxide upon a substrate or attack by external hydroxide upon metal-co-ordinated substrate. As we have shown above, both of these mechanisms are possible with non-labile model systems. 

Acidic proteinoids accelerate the hydrolysis of the unnatural substrate, p-nitrophenyl acetate 7,8). P-Nitrophenyl acetate has been used as a substrate for both natural esterases and esterase models. The imidazole ring of histidine is involved in the active site of a variety of enzymes, including hydrolytic enzymes. Histidine residues of proteinoid play a key role in the hydrolysis, the contribution to activity of residues of lysine and arginine is minor, and no activity is observed for proteinoid containing no basic amino acid 7). 

V. Seddou, 1. Hcttiing u 5 P. So niello. Hydrolytic enzyme production by Clostridium dfficile and its relationship to toxin production and virulence in the hamster model. 7. Med. Microbiol 37 169 (1990). 

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)... 

More detailed discussion of food polymers and their functionality in food is now difficult because of the lack of the information available on thermodynamic properties of biopolymer mixtures. So far, the phase behaviour of many important model systems remains unstudied. This particularly relates to systems containing (i) more than two biopolymers, (ii) mixtures containing denatured proteins, (iii) partially hydrolyzed proteins, (iv) soluble electrostatic protein-polysaccharide complexes and conjugates, (v) enzymes (proteolytic and amylolytic) and their partition coefficient between the phases of protein-polysaccharide mixtures, (vi) phase behaviour of hydrolytic enzyme-exopolysaccharide mixtures, exopolysaccharide-cell wall polysaccharide mixtures and exopolysaccharide-exudative polysaccharide mixtures, (vii) biopolymer solutes in the gel networks of one or several of them, (viii) enzymes in the gel of their substrates, (ix) virus-exopolysaccharide, virus-mucopolysaccharides and virus-exudative gum mixtures, and so on. 

Poly(amino acids) are attractive enzyme models because of their structural similarity. In fact, characteristic pH dependences of the cataljrtic rate were found and this was considered to reflect the conformational peculiarity of poly(amino acids). Unfortur nately, rate enhancements are only moderate and characterization of the catal3rtic site is difficult. Interesting results were obtained in the catalysis of oligopeptides, which supposedly mimic the active site of some hydrolytic enzymes. The stereoselectivity seems to be realized with oligopeptides more easily than with vinyl polymer catalysts. 

This complex has been shown to be an excellent structural and functional model for the zinc hydrolytic enzymes, particularly carbonic anhydrase but also carboxypeptidase and the zinc phosphate esterases (24-26). The same complex also catalyzes the hydration of acetaldehyde and hydrolysis of carboxylic esters. These reactions appear to progress via a mechanism similar to that proposed for carbonic anhydrase. The rates are slower for [Zn([12]aneN3)OH] than for the enzyme but an order of magnitude faster than for existing model systems such as [(NH3)5Co(OH)]2+ (26). 

Enzymatic assays can be applied in the marine environment to provide indirect information on dissolved compounds that are available to fuel bacterial production. Approaches that have been commonly appHed include measuring hydrolytic enzyme activities in seawater and monitoring degradation rates of model compounds. Protein hydrolysis in seawater is rapid as expressed by model protein studies (e.g., Nunn et al., 2003 Pantoja and Lee, 1999). This rapid and selective removal of dissolved proteins explains the relatively minor contribution from proteins to the accumulating DOM reservoir even though proteins are by far the most abundant intracellular biochemical. In an elegant study, Nunn and coworkers (2003) used matrix assisted laser desorption/ionization (MALDI) time of flight (TOP) mass... 

Hydrolytic enzymes that release two products in a defined sequence require a slightly more complicated model for their kinetic behavior. These enzymes pass through two intermediate stages in the catalytic cycle, E S and E S, and the formation of products involves two steps with associated rate constants k2 and k3 ... 

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