Enzyme-Catalyzed Cascade Reactions

Multi-Step Enzyme Catalysis Biotransformations and Chemomzymatic Synthesis Edited by Eduardo Garda-Junceda 

Copyright 2008 WILEY-VCH Verlag GmbH Co. KGaA, Weinheim ISBN 978-3-527-31921-3 

Strictly speaking a catalytic cascade process is one in which all of the catalysts (enzymes or chemocatalysts) are present in the reaction mixture from the outset. A one-pot process, on the other hand, is one in which several reactions are conducted sequentially in the same reaction vessel, without the isolation of intermediates. However, not all of the reactants or catalysts are necessarily present from the outset. Hence, a cascade process is by definition a one-pot process, but the converse is not necessarily true. Clearly a cascade process is a more elegant solution, but a one-pot process that is not, according to the strict definihon, a cascade reaction may have equal practical uhlity. In this chapter we shall be primarily concerned with enzymatic cascade processes, but the occasional chemocatalytic step may be included where relevant and sometimes a sequential one-pot procedure may slip through the net. 

As noted in the preceding section, successful compartmentahzation by immobilization may be the key to the compatibility of different enzymes in a cascade 

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Substrates or products of the TK reaction cannot be specifically quantified by any molecular property, which requires a subsequent specific chemical or enzyme-catalyzed cascade reaction to create a measurable signal for specific quantification. Such coupled assays can be classified according to the nature of the auxiliary agent involved in the cascade reaction. Depending on the principle of the assay, continuous measurements or only discontinuous end-point determinations can be made. 

Figure 24.28. A Higher Level in the Regulatory Cascade of Glutamine Synthetase. and Pq the regulatory proteins that control the specificity of adenylyl transferase, are interconvertible. P is converted into Pq by uridylylation, which is reversed by hydrolysis. The enzymes catalyzing these reactions are regulated by the concentrations of metabolic intermediates.

In 2012, Kroutil and co-workers reported the first amination reaction of primary alcohols with ammonium chloride by an artificial multi-enzyme-catalyzed cascade method (Scheme 29) [173]. The authors assumed that the reaction might proceed by two steps. Initially, the alcohol was oxidized by an alcohol dehydrogenase (ADH), consuming NAD" " and leading to the formation of the aldehyde and NADH. Then, the aldehyde intermediate was aminated with an amine donor L-alanine by a w-transaminase (w-TA). Finally, by combining ADH-hT (ADH from Bacillus stearothermophilus) with CV-w-TA (w-TA from Chromobacterium violaceuni), the amination of various primary alcohols successfully afforded the corresponding primary amines in 2-99 % yields. 

Members of the CHS/STS family of condensing enzymes are relatively modest-sized proteins of 40-47 kDa that function as homodimers. Each enzyme typically reacts with a cinnamoyl-CoA starter unit and catalyzes three successive chain extensions with reactive acetyl groups derived from enzyme catalyzed decarboxylation of malonyl-CoA.11 Release of the resultant tetraketide together with or prior to polyketide chain cyclization and/or decarboxylation yields chalcone or resveratrol (a stilbene). Notably, CHS and STS catalyze identical reactions up to the formation of the intermediate tetraketide. Divergence occurs during the termination step of the biosynthetic cascade as each tetraketide intermediate undergoes a distinct cyclization reaction (Fig. 12.2). 

Microbial biofuel cells were the earliest biofuel cell technology to be developed, as an alternative to conventional fuel cell technology. The concept and performance of several microbial biofuel cells have been summarized in recent review chapters." The most fuel-efficient way of utilizing complex fuels, such as carbohydrates, is by using microbial biofuel cells where the oxidation process involves a cascade of enzyme-catalyzed reactions. The two classical methods of operating the microbial fuel cells are (1) utilization of the electroactive metabolite produced by the fermentation of the fuel substrate " and (2) use of redox mediators to shuttle electrons from the metabolic pathway of the microorganism to the electrodes. ... 

The classical example is blood clotting, where successive steps involving enzyme-catalyzed proteolysis converts an inactive (or weakly active) proenzyme into its highly active form. Although unknown at the time of Wald s classical report, kinase-type and nucleotidyltransferase-type reactions (See Enzyme Cascade Kinetics) are frequently the source of biological signal transduction and amplification. 

The second stage of this cascade takes place at the cytoplasmic surface of the bacterial cell membrane. In the first reaction, MraY catalyzes a pyrophosphate exchange reaction wherein the UDP-MurNAc-pentapeptide precursor is coupled to a membrane-anchored C55 lipid carrier with ejection of UMP to provide undecaprenylpyrophosphoryl-MurNAc-pentapeptide 4, also know as lipid I. In the second reaction, the MurG enzyme catalyzes the transfer of GlcNAc from a UDP-GlcNAc precursor to the C(4)-hydroxyl group of the lipid-linked MurNAc-pentapeptide. The product of this enzymatic reaction, lipid II5, is the... 

Enzymes in the cross-linked crystal form are essentially impervious to degradation by exogenous proteases and from autolysis, in the case of CLCs of proteases themselves [5], This stability makes the enzyme-catalyzed preparation of peptides and peptide mimics truly practical [6], Examples will be discussed in more detail in Sec. IV. Further, one could conceive of using multiple enzymes in one-pot reaction systems mimicking natural biosynthetic cascades. Indeed, the application of this concept has been reported for a mixture of lipoamide dehydrogenase and lactate dehydrogenase [19],... 

Steroid biosynthesis occurs by enzyme-catalyzed epoxidation of squalene to 3deld squalene oxide, followed by acid-catalyzed cyclization and an extraordinary cascade of nine sequential carbocation reactions to yield lanosterol (Figure 27.6). Lanosterol is then degraded by other enzymes to... 

Amplification in a sequence of enzyme-catalyzed reactions suggests that more of the product is formed at each stage than in the previous stage. However, in the coagulation cascade the principal amplification is of reaction rate, not quantity of product formed. 

In nature a cascade of enzyme-catalyzed reactions is involved for the biosynthesis of starch. When selecting the appropriate enzymes and reaction circumstances reactions with multiple enzymes can be performed in vitro. Synthetic glycogen was first synthesized in vitro by Cori [146] in 1943 via the cooperative action of muscle phosphorylase and branching enzymes isolated from rat liver and rabbit heart. 

A second level of amplification is illustrated by the cAMP-mediated stimulation of glycogenolysis. As we just discussed, cAMP promotes glycogen degradation via a three-stage cascade, that is, a series of reactions in which the enzyme catalyzing one step is activated (or inhibited) by the product of a previous step (see Figure 13-17a). The amplification that occurs in such a cascade depends on the number of steps in it. 

In this chapter the arachidonate cascade will be reviewed from an enzymological point of view. Most steps of the cascade are enzyme-catalyzed reactions although some steps have only been implicated to involve enzymes. Several but not all enzymes have been purified and their properties extensively investigated the reaction mechanisms have been discussed in detail. A number of review articles dealing with PG biosynthesis and metabolism have been published [1-12] although only a few of these describe and discuss the current status of studies on the enzymes responsible for each of the individual steps of the araehidonate eascade. 

Further enhancement in detection sensitivity of reporter enzymes is achievable by enzyme amplification or cascade reactions often termed enzyme cycling assays. One approach is to use alkaline phosphatase as the reporter enzyme. Phosphatase cleavage of NADP forms NAD, which enters cyclic reactions catalyzed by alcohol dehydrogenase and diaphorase. Each turnover of phosphatase substrate initiates a cascade resulting in numerous detectable product molecules. Such approaches, perhaps incorporating chemiluminophores as the terminal product, hold promise for further extension of the sensitivity of immunoenzymatic methods. 

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