Enzyme-bound cofactor regeneration

Reactivation of UDP-Glc 4-epimerase can be achieved by incubation with the transition state analogs, dUDP- or dTDP-6-deoxy-4-ketoglucose (40, 37), resulting in an enzyme-bound cofactor regeneration (Fig. 30). The full activity of UDP-Glc 4-epimerase was recovered and the stability of the enzyme increased by incubation with these transition state analogs which have been synthesized by us on a 0.1 to 1 g scale [272,331,332] (see also Sections 6.1 and 6.2). With this... 

L-Amino acid transaminases are ubiquitous in nature and are involved, be it directly or indirectly, in the biosynthesis of most natural amino acids. All three common types of the enzyme, aspartate, aromatic, and branched chain transaminases require pyridoxal 5 -phosphate as cofactor, covalently bound to the enzyme through the formation of a Schiff base with the e-amino group of a lysine side chain. The reaction mechanism is well understood, with the enzyme shuttling between pyridoxal and pyridoxamine forms [39]. With broad substrate specificity and no requirement for external cofactor regeneration, transaminases have appropriate characteristics to function as commercial biocatalysts. The overall transformation is comprised of the transfer of an amino group from a donor, usually aspartic or glutamic acids, to an a-keto acid (Scheme 15). In most cases, the equilibrium constant is approximately 1. 

Some enzymes require nonprotein cofactor molecules for catalysis, as with NAD mentioned above. A cofactor may be covalently bound to the enzyme, such as FAD in the case of GOx, but others are diffusing freely in solution. Figure 9.3 shows the mechanism of aldehyde dehydrogenase (ALDH), in which an aldehyde is oxidized to a carboxylic acid in conjunction with an NAD cofactor [28]. The NAD cofactor simultaneously binds in the enzyme active site and is released as the reduced form NADH. In this case, NAD acts as the oxidizing substrate of the enzyme and is regenerated back to NAD elsewhere in the system, independently of ALDH. Thus, NAD and other freely diffusing redox cofactors may be thought of as natural mediators. Pyrroloquinohne quinone (PQQ) is another frequently encountered redox cofactor. 

However, when the cofactor is bound strongly to the enzyme as a prosthetic group, cofactor addition and additional enzymatic processes for cofactor regeneration are not needed. A representative example for such enzymes is amino add oxidases that catalyze the oxidation of amino adds to a-keto acids under simultaneous reduction of molecular oxygen to hydrogen peroxide. 

A lot of analytical techniques have been proposed in recent decades and most of them are based on enzymes, called dehydrogenases, which are not sensitive to oxygen and need cofactors such as NAD". The key problems which seriously hamper a wide commercialization of biosensors and enzymatic kits based on NAD-dependent enzymes are necessity to add exogenous cofactor (NAD" ) into the samples to be analyzed to incorporate into the biologically active membrane of sensors covalently bounded NAD" to supply the analytical technique by NAD -regeneration systems. 

In the field of enzymatic oxidations especially, the class of flavoenzymes with bound FAD as cofactor is interesting for synthetic applications. The regeneration of the oxidized FAD within the enzyme can be performed by oxygen. In this case, however, hydrogen peroxide is formed, which drastically diminishes the enzyme stability and activity, rendering it unsuitable for synthesis. 

Despite the similarities in the requirements for cleavage of SAM in these systems, LAM—and presumably all enzymes that will be in its subclass—displays one distinct difference. The cleavage of the cofactor is a reversible process. In fact, at the end of each turnover the cofactor would have to be regenerated to allow the product to be released and a new substrate molecule to be bound. This is necessitated by the instability of the 5 -deoxyadenosyl 5 -radical which, unlike the... 

C. regenerate the oxidized lipoic acid cofactor bound to one of the other enzymes in the complex... 

In a separate pathway, MTP synthase is regenerated (grouped within a grey box in Fig. 4.12) by transferring sulfur to the small subunit. In Escherichia coli, MoeB catalyses the adenylation of the carboxyl of the C-terminal Gly residue of MoaD, in a reaction very similar to that catalysed by the El enzyme that activates ubiquitin in the ubiquitin/proteasome pathway referred to earlier. The AMP-activated MoaD is then sulfurated by sulfide transfer from an unknown donor. It is assumed that Cu is inserted directly after dithiolate formation. In the last two steps, adenylated MPT is formed from Mg-ATP, and, finally, in the presence of molybdate (MoOa ), bound MPT-AMP is hydrolysed, Cu is released and Mo is transferred to the MPT dithiolate, and the MoCo cofactor is released. 

Scheme 8.1-1 The regeneration of protein-lipoates cofactors and enzyme thiols bound to arsenic by reaction with small dithiols.

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