Catalytic reactions enzyme-catalysed

Most reactions in cells are carried out by enzymes [1], In many instances the rates of enzyme-catalysed reactions are enhanced by a factor of a million. A significantly large fraction of all known enzymes are proteins which are made from twenty naturally occurring amino acids. The amino acids are linked by peptide bonds to fonn polypeptide chains. The primary sequence of a protein specifies the linear order in which the amino acids are linked. To carry out the catalytic activity the linear sequence has to fold to a well defined tliree-dimensional (3D) stmcture. In cells only a relatively small fraction of proteins require assistance from chaperones (helper proteins) [2]. Even in the complicated cellular environment most proteins fold spontaneously upon synthesis. The detennination of the 3D folded stmcture from the one-dimensional primary sequence is the most popular protein folding problem. 

The specificity of enzyme reactions can be altered by varying the solvent system. For example, the addition of water-miscible organic co-solvents may improve the selectivity of hydrolase enzymes. Medium engineering is also important for synthetic reactions performed in pure organic solvents. In such cases, the selectivity of the reaction may depend on the organic solvent used. In non-aqueous solvents, hydrolytic enzymes catalyse the reverse reaction, ie the synthesis of esters and amides. The problem here is the low activity (catalytic power) of many hydrolases in organic solvents, and the unpredictable effects of the amount of water and type of solvent on the rate and selectivity. 

However, there are disadvantages to using immobilised cells. The cell may contain numerous catalytically active enzymes, which may catalyse unwanted side reactions. Also, the cell membrane itself may serve as a diffusion barrier, and may reduce productivity. The matrix may sharply reduce productivity if the microorganism is sensitive to product inhibition. One of the disadvantages of immobilised cell reactors is that the physiological state of the microorganism cannot be controlled. 

This is the first example of a reaction for which the presence of a chelating ligand was observed to facilitate rather than retard metal-catalysed epoxidation (Gao et al., 1987). It was found that the use of molecular sieves greatly improves this process by removing minute amounts of water present in the reaction medium. Water was found to deactivate the catalyst. All these developments led to an improved catalytic version that allows a five-fold increased substrate concentration relative to the stoichiometric method. Sensitive water-soluble, optically active glycidols can be prepared in an efficient manner by an in situ derivatisation. This epoxidation method appears to be competitive with enzyme-catalysed processes and was applied in 1981 for the commercial production of the gypsy moth pheromone, (-1-) disparlure, used for insect control (Eqn. (25)). 

Each enzyme has a working name, a specific name in relation to the enzyme action and a code of four numbers the first indicates the type of catalysed reaction the second and third, the sub- and sub-subclass of reaction and the fourth indentifies the enzyme [18]. In all relevant studies, it is necessary to state the source of the enzyme, the physical state of drying (lyophilized or air-dried), the purity and the catalytic activity. The main parameter, from an analytical viewpoint is the catalytic activity which is expressed in the enzyme Unit (U) or in katal. One U corresponds to the amount of enzyme that catalyzes the conversion of one micromole of substrate per minute whereas one katal (SI unit) is the amount of enzyme that converts 1 mole of substrate per second. The activity of the enzyme toward a specific reaction is evaluated by the rate of the catalytic reaction using the Michaelis-Menten equation V0 = Vmax[S]/([S] + kM) where V0 is the initial rate of the reaction, defined as the activity Vmax is the maximum rate, [S] the concentration of substrate and KM the Michaelis constant which give the relative enzyme-substrate affinity. 

Finally, the most complex synthetic reaction clearly catalysed by RNA molecules generated by in vitro selection is the formation of the C-N bond of a nucleoside (Scheme 7), from 4-thiouracil and most of the natural substrate for the natural (uracil phos-phoribotransferase) reaction.1461. (Thiouracil was used because it is easily tagged by alkylation on sulfur.) The catalytic RNAs produced by 11 rounds of selection required Mg++ cations and had kcat as high as 0.13 min-1,with kcaJKM at least 107 times greater than the (undetectable) uncatalyzed reaction. Once again these systems are convincing, rather efficient enzyme mimics. 

The first zinc enzyme to be discovered was carbonic anhydrase in 1940, followed by car-boxypeptidase A some 14 years later. They both represent the archetype of mono-zinc enzymes, with a central catalytically active Zn2+ atom bound to three protein ligands, and the fourth site occupied by a water molecule. Yet, despite the overall similarity of catalytic zinc sites with regard to their common tetrahedral [(XYZ)Zn2+-OH2] structure, these mononuclear zinc enzymes catalyse a wide variety of reactions, as pointed out above. The mechanism of action of the majority of zinc enzymes centres around the zinc-bound water molecule,... 

Schultz and coworkers (Jackson et a ., 1988) have generated an antibody which exhibits behaviour similar to the enzyme chorismate mutase. The enzyme catalyses the conversion of chorismate [49] to prephenate [50] as part of the shikimate pathway for the biosynthesis of aromatic amino acids in plants and micro-organisms (Haslam, 1974 Dixon and Webb, 1979). It is unusual for an enzyme in that it does not seem to employ acid-base chemistry, nucleophilic or electrophilic catalysis, metal ions, or redox chemistry. Rather, it binds the substrate and forces it into the appropriate conformation for reaction and stabilizes the transition state, without using distinct catalytic groups. 

As far as the use of ferrocene molecules as amperometric sensors is concerned, they have found wide use as redox mediators in the so-called enzymatic electrodes, or biosensors. These are systems able to determine, in a simple and rapid way, the concentration of substances of clinical and physiological interest. The methodology exploits the fact that, in the presence of enzyme-catalysed reactions, the electrode currents are considerably amplified.61 Essentially it is an application of the mechanism of catalytic regeneration of the reagent following a reversible charge transfer , examined in detail in Chapter 2, Section 1.4.2.5 ... 

The enzyme adsorbed on the electrode showed large IT2 oxidation currents, with an activity greater than the catalytic activity with electron acceptors and donors (Pershad et al. 1999). This demonstrates that the electron transfer to [NiFe] active site, and reaction with IT2 are extremely efficient, and the factor that limits the enzyme-catalysed rate is the diffusion of the electron donors and acceptors to the enzyme. 

The best way to combine all these parameters is to trace back the catalytic action of enzymes to intramolecularity. It is generally accepted that when van der Waals distances (contact distances) are imposed for definite times upon reactive groups, intramolecular reactions occur then at enzyme-like rates (accelerations of 10 to 10 0 are associated to enzyme-catalysed reactions). On the other hand, according to the Page-Jencks theory [17] the fast rates of intramolecular reaction "are merely an entropic consequence of converting a bimolecular reaction into a unimolecular reaction". 

Enzymes provide catalytic pathways which are often superior to non-enzymic routes. The advantage of enzymes can overcome their disadvantages as relatively unstable species which workbest in aqueous solution. In synthetic processes, the selectivity of the enzyme-catalysed process is not approached by chemical catalysis particularly regarding the enantioselective synthesis of chiral molecules. Furthermore, protective group chemistry is often not required in enzyme-catalysed reactions. 

Uni-site and multi-site catalyses were essentially confirmed with E. coli Fi, although the ratio of the rates of multi- to uni-site hydrolysis with this enzyme was slightly lower than beef heart Fi.44 47 48 The mutation with the a or 0 subunit defective only in multi-site catalysis was isolated. Typically, aSer-373—Phe or aArg-376—Cys mutant enzyme with amino acid substitution in the a subunit had low multi-site activity (less than 0.1% that of the wild type), but essentially normal uni-site activity.49 50 These mutant enzymes were defective in catalytic cooperativity, but normal in the catalytic reaction in a single catalytic... 

Generally it is possible to activate enzymes under pressure, but at high hydrostatic pressure a decrease in activity occures due to pressure induced inactivation. The impact of pressure on the reaction rates is quite complex because of the different susceptibilities of the catalytic steps. It should be useful to perform enzyme catalysed reactions under hydrostatic pressure because pressure and temperature can have contrary effects. 

At temperatures greater than 45 C, thermal denaturation of the enzyme becomes increasingly significant. At about 55 C, rapid denaturation completely destroys the catalytic function of the enzyme protein. The effect of temperature on the rates of enzyme-catalysed reactions is illustrated in the figure above. 

Chorismate mutase provides an example of an enzyme where QM/MM calculations have identified an important catalytic principle at work [8], This enzyme catalyses the Claisen rearrangement of chorismate to prephenate. The reaction within the enzyme is not believed to involve chemical catalysis, and this pericylic reaction also occurs readily in solution. Lyne et al. [8] investigated the reaction in chorismate mutase in QM/MM calculations, at the AMI QM level (AMI was found to perform acceptably well for this reaction in comparisons with ab initio results for the reaction in the gas phase [8]). Different sizes of QM system were tested in the QM/MM studies (e.g. including the substrate and no, or up to three, protein side chains), and similar results found in all cases. The reaction was modelled by minimization along an approximate reaction coordinate, defined as the ratio of the forming C-C and breaking C-0 bonds. Values of the reaction coordinate were taken from the AMI intrinsic reaction coordinate for the gas-phase reaction. 

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