Enzyme catalysed

A mild and inexpensive way to reduce aldehydes or ketones uses fermenting Baker s yeast. This is a whole-cell system that contains oxidoreductase enzymes and cofactors that reduce the substrate. The ketonic carbonyl groups of (3-keto-esters and cyclic ketones are reduced with high selectivity using Baker s yeast. Typical in this regard is the reduction of ethyl acetoacetate, which gives ethyl 3-hydroxybutyrate as predominantly the (5)-stereoisomer (7.101). Similarly, the ketone 114 gave the optically active 3-hydroxyproline derivative 115 (7.102). 

Interestingly, reduction of ethyl 4-chloroacetoacetate with Baker s yeast gave predominantly the corresponding (5)-alcohol (i.e. the opposite configuration from that of the alcohol from ethyl acetoacetate itself) (7.103), but the corresponding octyl ester gave almost entirely the (/f)-alcohol. The stereochemistry of the reduction depends on the shape of the molecule and it is likely that the yeast contains at least two different oxidoreductase enzymes which produce the two enantiomeric alcohols at different rates. 

in Comprehensive Organic Synthesis, ed. B. M. Trost and I. Heming, vol. 8 (Oxford Pergamon Press, 1991),p. 183 E. Santaniello, P. Ferraboschi, P. GrisentiandA. Manzocchi, Cftem./f v.,92(1992), 1071  

Nakamura, R. Yamanaka, T. Matsuda and T. Harada, Tetrahedron Asymmetry, 14 (2003), 2659. 

Reduction with isolated enzymes avoids difficulties associated with diffusion limitations and also avoids the presence of many different enzymes, present in the whole cell, which can cause side reactions or reduced enantioselectivity. The main drawback, however, is the instability of the isolated enzyme and the requirement for added co-factor NAD(H) or NADP(H), which are the oxidized (or reduced) forms of nicotinamide adenine diphosphate or its 2 -phosphate derivative. These co-factors are expensive, but can be used as catalysts in the presence of a co-reductant such as formate ion HCOO or an alcohol (e.g. isopropanol or ethanol). The reduction of ketones occurs by transfer of hydride from the C-4 position of the dihydropyridine ring of NADH or NADPH (7.105). Only one of the two hydrogen atoms is transferred and this process occurs within the active site of the enzyme to promote asymmetric reduction. 

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Oxidoreduciases. Enzymes catalysing redox reactions. The substrate which is oxidized is regarded as the hydrogen donor. This group includes the trivially named enzymes, dehydrogenases, oxidases, reductases, peroxidases, hydrogenases and hydroxylases. 

Hydrolases. Enzymes catalysing the hydrolytic cleavage ofC —O, C —N and C —C bonds. The systematic name always includes hydrolase but the recommended name is often formed by the addition of ase to the substrate. Examples are esterases, glucosidases, peptidases, proteinases, phospholipases. Other bonds may be cleaved besides those cited, e.g. during the action of sulphatases and phosphatases. 

Ligases (syniheiases). Enzymes catalysing the joining together of two molecules coupled with the hydrolysis of a pyrophosphate bond in ADP or a similar triphosphate. They include some carboxylases and many enzymes known as synthetases. 

Often there is more than one enzyme catalysing the same reaction. 

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. 

In the following pages an account is given of some of the more simple reactions which enzymes catalyse. The reactions have been selected partly because they are of particular interest to the organic chemist, and partly because they are capable of simple and ready demonstration in the laboratory. 

The accompanying table gives details of a few of the simpler reactions which enzymes catalyse. Those for which practical directions are given in the following pages arc marked with an asterisk. 

An enzyme-amplified detection scheme, based on tire coupling of a streptavidin-alkaline phosphatase conjugate and biotinylated target sequences was then applied. The enzyme catalysed the hydrolysis of the elecn oiiractive a-naphthyl phosphate to a-naphtlrol this product is elecU oactive and has been detected by means of differential... 

J. W. Cornforth (Sussex) stereochemistry of enzyme-catalysed reactions. 

Table 2.1 Some reactions catalysed by microbial enzymes. In principle each enzyme catalyses the reverse process as well.

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. 

An alkaline pH (- pH 11) is desirable in order to achieve high conversion rates increase solubility of L-phenylalanine inhibit enzymes catalysing degradation of L-phenylalanine and formation of byproducts reduce inhibition of the reaction by the keto form of phenylpyruvic arid. 

Most enzymes catalyse reactions and follow Michaelis-Menten kinetics. The rate can be described on the basis of the concentration of the substrate and the enzymes. For a single enzyme and single substrate, the rate equation is ... 

In general, enzymes are proteins and cany charges the perfect assumption for enzyme reactions would be multiple active sites for binding substrates with a strong affinity to hold on to substrate. In an enzyme mechanism, the second substrate molecule can bind to the enzyme as well, which is based on the free sites available in the dimensional structure of the enzyme. Sometimes large amounts of substrate cause the enzyme-catalysed reaction to diminish such a phenomenon is known as inhibition. It is good to concentrate on reaction mechanisms and define how the enzyme reaction may proceed in the presence of two different substrates. The reaction mechanisms with rate constants are defined as ... 

The role of divalent cations in the mechanism of enzyme catalysed phosphoryl and nucleotidyl transfer reactions. A. S. Mildvan and C. M. Grisham, Struct. Bonding (Berlin), 1974,20,1-21 (88),... 

Gani D, Wilkie J (1997) Metal Ions in the Mechanism of Enzyme Catalysed Phosphate Monoester Hydrolyses. 89 133-176... 

Transition state theory has been useful in providing a rationale for the so-called kinetic isotope effect. The kinetic isotope effect is used by enzy-mologists to probe various aspects of mechanism. Importantly, measured kinetic isotope effects have also been used to monitor if non-classical behaviour is a feature of enzyme-catalysed hydrogen transfer reactions. The kinetic isotope effect arises because of the differential reactivity of, for example, a C-H (protium), a C-D (deuterium) and a C-T (tritium) bond. 

Such a subunit structure permits the construction of the virus partieles by a proeess in which the subunits self-assemble into structures held together by non-eovalent intermolecular forces as occurs in the process of erystallization. This eliminates the need for a sequenee of enzyme-catalysed reactions for coat synthesis. It also provides an automatic quality-control system, as subunits which may have major stmctural defects fail to become ineorporated into complete partieles. 

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