Consecutive enzymatic reactions

Ruiz, S., Feliu, ).A., Caminal, G., Alvaro, G., and LopezSantin,). (1997) Reaction engineering for consecutive enzymatic reactions in peptide synthesis application to the synthesis of a pentapeptide. Biotechnol. Prog., 13 (6), 783-787. 

In a living system, the transformation of a substance is catalyzed by its recognized enzyme. Therefore, all natural compounds and their analogues can, in principle, be prepared by a sole enzymatic reaction or by consecutive enzymatic reactions along the metabolic pathway in vitro or in vivo. 

Fig. 9. Consecutive enzymatic reactions for synthesis of 3-cyclohexylpropyl caffeate from 5-caffeoylquinic acid. In all reaction steps, [bmim][NTf2] was used as the solvent. Compound 1. 5-caffeoylquinic acid, 2. methyl caffeate, 3 3-cydohexyl-l-propanol, and 4 3-cyclohexylpropyl caffeate.

Consecutive enzymatic reactions for synthesis of 3-cyciohexyipropyi caffeate... 

The authors chose pyruvic acid as their model compound this C3 molecule plays a central role in the metabolism of living cells. It was recently synthesized for the first time under hydrothermal conditions (Cody et al., 2000). Hazen and Deamer carried out their experiments at pressures and temperatures similar to those in hydrothermal systems (but not chosen to simulate such systems). The non-enzymatic reactions, which took place in relatively concentrated aqueous solutions, were intended to identify the subsequent self-selection and self-organisation potential of prebiotic molecular species. A considerable series of complex organic molecules was tentatively identified, such as methoxy- or methyl-substituted methyl benzoates or 2, 3, 4-trimethyl-2-cyclopenten-l-one, to name only a few. In particular, polymerisation products of pyruvic acid, and products of consecutive reactions such as decarboxylation and cycloaddition, were observed the expected tar fraction was not found, but water-soluble components were found as well as a chloroform-soluble fraction. The latter showed similarities to chloroform-soluble compounds from the Murchison carbonaceous chondrite (Hazen and Deamer, 2007). 

It shall be assumed that most biosensors are designed on the basis of enzymatic working elements. In fact, two consecutive reactions proceed in such electrochemical systems first is the enzymatic reaction, which generates electrochemically active compounds to the system this compound acts as an intermediate or a final product of the reaction (a mediate). These compounds then enter the electron transfer reaction with conducting material. 

Fig. 24.8. Computational simulation analysis of conformational dynamics in T4 lysozyme enzymatic reaction, (a) Histograms of fopen calculated from a simulated single-molecule conformational change trajectory, assuming a multiple consecutive Poisson rate processes representing multiple ramdom walk steps, (b) Two-dimensional joint probability distributions <5 (tj, Tj+i) of adjacent pair fopen times. The distribution <5(ri, Ti+i) shows clearly a characteristic diagonal feature of memory effect in the topen, reflecting that a long topen time tends to be followed by a long one and a short fopen time tends to be followed by a short one...

As discussed above, an enzymatic reaction is usually found to be more rapid in one direction than the other so that the reaction is virtually irreversible.If the product of the reaction in one direction is removed as it is formed (Le., because it is the substrate of a second enzyme present in the reaction mixture), the equilibrium of the first enzymatic process is displaced so that the reaction may continue to completion in that direction. Reaction sequences in which the product of one enzyme-catalyzed reaction becomes the substrate of another enzyme, often through many stages, are characteristic of metabolic processes. Analytically, several enzymatic reactions also may be Unked together to provide a means of measuring the activity of the first enzyme or the concentration of the initial substrate in the chain. For example, the activity of creatine kinase is usually measured by a series of linked reactions, and glucose can be determined by consecutive reactions catalyzed by hexokinase and glucose-6-phosphate dehydrogenase. 

Hydrolysis now has been completed successfully by proteolytic enzymes from bacteria and moulds. In these reactions there are no problematic side products because they operate under mild conditions as mentioned above. The further processing after enzymatic hydrolysis is similar to the classically hydrolyzed products by heating and consecutive Maillard reaction. In the heating step the enzymes are denatured and destroyed [5]. 

Realizing hybrid approaches by coupling either a reactor or separation unit with a membrane is another very interesting possibility [50, 51]. There are two options in which membranes could be used coupled to reactors. The first is to distribute the feed of one of the reactants to a packed bed of catalyst, and thus realize a better profile of concentration along the reactor to minimize hot spots and consecutive reactions. This approach has resulted in increased selectivity in partial oxidation reactions. The second approach is to either remove a product that inhibits a reaction (e.g., in enzymatic reactions) or to remove a product to shift the equilibrium. Examples are in the continuous removal of water in dehydration reactions, or of H2 in dehydrogenation reactions. 

Cyanopyridine generated by the ammoxidation is hydrolyzed using an enzymatic catalyst with practically quantitative yields. This efficient procedure avoids the consecutive hydrolysis reaction to nicotinic acid (here a by-product ). 

Structure of the reaction system. The term structure should include the sum of the chemical reactions occurring within the reaction system, e.g. parallel reactions, consecutive reactions (see Eq. (4)), coupled reactions such as in the case of coenzyme regeneration (see Eq. (49)), non-catalyzed reactions occurring alongside the enzymatic reaction. 

Figure 7 Eight ALA molecules are converted into uroporphyrinogen III by three consecutive enzymatic steps via porphobilinogen and preuroporphyrinogen. These reactions are catalyzed by PBGS, PBGD, and UROS. A = acetate, P = propionate.

Figure 11 Uroporphyrinogen III is converted into protoheme by four consecutive enzymatic steps. Decarboxylation of uroporphyrinogen III gives rise to coproporphyrinogen I which in turn is oxidatively decarboxylated to protoporphyrinogen IX. Oxidation of protoporphyrinogen IX produces protoporphyrin IX and final iron insertion gives rise to protoheme. These four reactions steps are catalyzed by UROD, OPO, PPO, and FO, respectively.

The type of reaction which is probably of most importance in the enzymatic degradation of polymers is the bimolecular reaction illustrated above, in which the enzyme catalyzes the interaction of the polymer and a low molecular reagent (such as water in a hydrolysis reaction). These reactions can occur by either a single displacement or a double displacement mechanism. In the former, both substrates, A and B below, are bound to the enzyme by consecutive, reversible reactions, after which the final complex, EAB, dissociates into the products, C and D, and the free enzyme, as follows ... 

Minimal motion rule The observed stereochemistry of enzymatic reactions usually assumes that the proposed intermediates remain oriented through consecutive steps in a minimal motion relationship to the interacting groups. The selection for minimal motion can be explained ... 

Since enzymes generally function under the same or similar conditions, several biocatalytic reactions can be carried out in a reaction cascade in a single flask. Thus, sequential reactions are feasible by using multienzyme systems in order to simplify reaction processes, in particular if the isolation of an unstable intermediate can be omitted. Furthermore, an unfavorable equilibrium can be shifted towards the desired product by linking consecutive enzymatic steps. This unique potential of enzymes is increasingly being recognized as documented by the development of multienzyme systems, also denoted as artificial metabolism [15]. 

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