Artificial hydrolytic enzymes

Although hydrolytic enzymes, esterases and amidases, are named after their major substrates, the same enzyme can often hydrolyze esters, thioesters, and amides therefore, the differentiation between esterases and amidases is sometimes artificial. The highest hydrolytic activity is in the liver, but the enzyme pseudocholinesterase is found in the serum. Gut bacteria also contain hydrolytic enzymes. 

The natural substrates for many enzymes do not show any significant spectral properties and alternative artificial substrates (analogues) may be used. Many hydrolytic enzymes can most easily be detected by using substrate analogues the glycosidases, for instance, may be measured using p-nitro-phenyl derivatives of the appropriate carbohydrates. Hydrolysis results in the production of p-nitrophenol, which can be measured at 404 nm. 

Rates of supply may be controlled, in part, by the activity of hydrolytic enzymes that degrade combined molecules in dissolved or particulate organic matter to monomeric constituents prior to enzymatic transport into bacterial cells (Skoog et al., 1999 see also Chapter 13). At present, in situ rates of such exoenzymes have not been accurately measured, but enzyme potential has been estimated with the addition of artificial substrates in saturating... 

To create artificial enzymes that could bind substrates in water solution with defined geometry, we examined dimers of cyclodextrins. As mentioned above, we used such dimers in mimics of hydrolytic enzymes [119, 120]. Now we wished to use them for mimics of cytochrome P-450. 

In some cases, mainly in the study of hydrolytic enzymes, the natural substrate must be replaced by an artificial one, that is chromolytic, chromogenic, chemiluminiscent,bioluminiscent, or fluorescent. 

Chitosan is a 3-1,4-linked glucosamine polymer and partially deacetylated form of chitin. Naturally, it is present as a major component of the Zygomycete cell walls and usually obtained by artificial deacetylation of chitin. Chitosanase (EC 3.2.1.99) is a hydrolytic enzyme which catalyzes the breakdown of chitosan into glucosamine oligomers (1). It has been isolated from several microorganisms inchidmg Streptomyces (2), Pseudomonas (3), Bacillus (4), Penicillium (5) and Fusarium (6). 

Metallomicelles made up of ligand surfactants and bound metal ions have been studied extensively as artificial hydrolytic metallo-enzymes. Many such systems have become large and complex, but a study of a simple (1 1) Co(II) complex with triethanolamine showed that in the presence of CTAB or Triton X-100, it could catalyse PNPP (67) hydrolysis at pH 7 with a rate enhancement factor of 1000-fold. " ... 

Enzymatic ester hydrolysis is a common and widespread biochemical reaction. Since simple procedures are available to follow the kinetics of hydrolytic reactions, great efforts have been made during the last years to explain this form of catalysis in chemical terms, i.e., in analogy to known non-enzymatic reactions, and to define the components of the active sites. The ultimate aim of this research is the synthesis of an artificial enzyme with the same substrate specificity and comparable speeds of reaction as the natural catalyst. 

Esters can be cleaved by template catalysts that use a metal ion as both a binding group and part of the catalytic system [79-81]. However, metal ion catalysis has also been extended to cases in which the principal substrate binding involves cyclodextrin inclusion indeed, the first catalyst described as an artificial enzyme was. such an example [82]. A cyclodextrin dimer 47 with a bound metal ion between the two cyclodextrins is a particularly effective hydrolytic catalyst for esters that can bind into both cyclodextrin units (Scheme 6-20) [83, 84]. 

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