Cofactor-Independent with Activated Substrates

In a related fashion, asymmetric amination of ( )-cinnamic acid yields L-phenylalanine using L-phenylalanine ammonia lyase [EC 4,3,1,5] at a capacity of 10,000 t/year [1274, 1601], A fascinating variant of this biotransformation consists in the use of phenylalanine aminomutase from Taxus chinensis (yew tree), which interconverts ot- to p-phenylalanine in the biochemical route leading to the side chain of taxol [1602], In contrast to the majority of the cofactor-independent C-0 and C-N lyases discussed above, its activity depends on the protein-derived internal cofactor 5-methylene-3,5-dihydroimidazol-4-one (MIO) [1603], Since the reversible a,p-isomerization proceeds via ( )-cinnamic acid as achiral intermediate, the latter can be used as substrate for the amination reaction. Most remarkably, the ratio of a- vs, 3-amino acid produced (which is 1 1 for the natural substrate, R = H) strongly depends on the type and the position of substituents on the aryl moiety While o-substituents favor the formation of a-phenylalanine derivatives, / -substituted substrates predominantly lead to p-amino analogs, A gradual switch between both pathways occurred with m-substituted compounds. With few exceptions, the stereoselectivity remained exceUent (Scheme 2,215) [1604, 1605],... 

Decarboxylation is one of the most common processes in natural metabolism. All decarboxylases [EC 4.1.1.-] cleave a substrate carboxylic group with or without the requirement of an enzymatic cofactor. There are three known decarboxylase types (i) thiamine diphosphate (ThDP)-dependent decarboxylases, (ii) pyridoxal phosphate (PLP)-dependent decarboxylases, and (iii) cofactor-independent decarboxylases (Figure 3.1) [1-4]. Cofactor-independent decarboxylases are specific for activated substrates. 

P-Gal has a molecular weight of 540,000 and is composed of four identical subunits of MW 135,000, each with an independent active site (Melchers and Messer, 1973). The enzyme has divalent metals as cofactors, with chelated Mg+2 ions required to maintain active site conformation. The presence of NaCl or dilute solutions (5 percent) of low-molecular-weight alcohols (methanol, ethanol, etc.) causes enhanced substrate turnover. P-Gal contains numerous sulfhy-dryl groups and is glycosylated. 

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. 

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