Cofactors for catalysis

Inspired by enzymatic mechanisms, several groups have attempted to obtain amidase antibodies by recruiting a metal cofactor for catalysis. Introduction of... 

Unlike S-COMT from rat liver, COMT (alfalfa) is also presumed to utilize a general base without the aid of any metal cofactor for catalysis. Specifically, methyl transfer from SAM (115) to caffeic acid (5) and/or... 

Many enzymes require cofactors for catalysis. Coenzymes are organic cofactors and derivatives of B vitamers. NAD" and FAD are well-known cofactors for dehydrogenases and oxidases, respectively. Many metal ions are inorganic cofactors. 

Many redox enzymes used in BFCs require cofactors for catalysis. For example, yeast alcohol dehydrogenase catalyzes the oxidation of ethanol to acetaldehyde, with the concomitant reduction of NAD" to NADH. Most commonly, cofactor specificity has been engineered to increase the activity of NADP(H)-dependent enzymes with NAD(H) or to correct a cofactor imbalance in a metabolic process. As the NAD(H) cofactor is more stable and less expensive than NADP(H), this generally improves the economics of a process [42—44]. 

There have been few reports in the literature of using non-natural cofactors for catalysis. Notably, in 2002, Lo and Fish synthesized a variety of /V-benzylnicotinamide derivatives with the goal of improving turnover and cofactor stability in chiral synthesis applications [45]. Whereas the wild-type enzymes tested displayed poor turnover with the cofactor derivatives, a series of engineered mutants with altered cofactor specificity were able to use the novel cofactors at rates approaching those of the natural cofactors [46,47]. 

For more than 20 years EHs have proved to be outstanding biocatalysts in a large number of applications in fine chemistry. This success is largely due to the fact that EHs are robust enzymes, which do not need any cofactors for catalysis. Furthermore,... 

Cofactors serve functions similar to those of prosthetic groups but bind in a transient, dissociable manner either to the enzyme or to a substrate such as ATP. Unlike the stably associated prosthetic groups, cofactors therefore must be present in the medium surrounding the enzyme for catalysis to occur. The most common cofactors also are metal ions. Enzymes that require a metal ion cofactor are termed metal-activated enzymes to distinguish them from the metalloenzymes for which metal ions serve as prosthetic groups. 

All characterized BVMOs contain a flavin cofactor that is crucial for catalysis while NADH or NADPH is needed as electron donor. An interesting observation is the fact that most reported BVMOs are soluble proteins. This is in contrast to many other monooxygenase systems that often are found to be membrane-bound or membrane-associated. In 1997, Willetts concluded from careful inspection of... 

All soil metabolic proce.sses are driven by enzymes. The main sources of enzymes in soil are roots, animals, and microorganisms the last are considered to be the most important (49). Once enzymes are produced and excreted from microbial cells or from root cells, they face harsh conditions most may be rapidly decomposed by organisms (50), part may be adsorbed onto soil organomineral colloids and possibly protected against microbial degradation (51), and a minor portion may stand active in soil solution (52). The fraction of extracellular enzyme activity of soil, which is not denaturated and/or inactivated through interactions with soil fabric (51), is called naturally stabilized or immobilized. Moreover, it has been hypothesized that immobilized enzymes have a peculiar behavior, for they might not require cofactors for their catalysis. 

Spin labeled 5 -deoxyadenosylcobinamide has been used as a cofactor for ethanolamine-ammonia-lyase and the ESR spectrum followed during catalysis (123). This spin labeled coenzyme is biologically active in this enzyme. Enzyme kinetics showed this derivative to have the same Vmax as the cofactor 5 -deoxyadenosylcobinamide, but it has a higher Km value of 5.1 X 10-6 M compared to 4.6 X 10-6 for 5 -deoxyadenosylcobinamide (123). When the spin labeled coenzyme was incubated with apoenzyme to give the enzyme-coenzyme complex, the nitroxide ESR spectrum resembled that of free spin label but the lines are broadened significantly. 

Many NRPs such as cyclosporin, complestatin, actinomycin, and chondramide contain N-methyl amides. M-Methyl transferase (N-MT) domains utilize S-adenosylmethionine (SAM) as a cofactor to catalyze the transfer of the methyl group from SAM to the a-amine of an aminoacyl-S-PCP substrate. The presence of M-methylamides in NRPs is believed to protect the peptide from proteolysis. Interestingly, N-MT domains are incorporated into the A domains of C-A-MT-PCP modules, between two of the core motifs (A8 and A9). MT domains contain three sequence motifs important for catalysis. ° 0-Methyl transferase domains are also found in NRPSs and likewise use the SAM cofactor. For instance, cryptophycin and anabaenopeptilide synthetases contain 0-MT domains for the methylation of tyrosine side chains. These 0-MT domains lack one of the three core motifs described for N-MT domains. ... 

Figure 11.1. Structures of cofactors required for catalysis of one-carbon reactions in methanogenic pathways.

So far 18 different members of HDACs have been discovered in humans and classified into four classes based on their homology to yeast histone deacetylases [33]. Class I includes four different subtypes (HDACl, 2, 3, 8), class II contains six subtypes tvhich are divided into two subclasses class Ila with subtypes HDAC4, 5, 7, 9 and class Ilb with HDAC6, 10. Class I and class II HDAC share significant structural homology, especially within the highly conserved catalytic domains. HDACs 6 and 10 are unique as they have two catalytic domains. HDACll is referred to as class IV. While the activity of class I, II and IV HDACs depends on a zinc based catalysis mechanism, the class III enzymes, also called sirtuins, require nicotinamide adenine dinucleotide as a cofactor for their catalysis. 

Amino acids important in cofactor and catalysis in human 1 lb-hydroxysteroid dehydrogenase types 1 and 2. (a) 1 lb-HSD type 1. Preference of 1 lb-HSD type 1 for NADPH resides in lysine-44 and arginine-66, which have positively charged side chains that stabilize the binding of the 2 -phosphate on NADPH. These residues also counteract the repulsive interaction between glutamic acid 69 and the phosphate group,... 

All adenosine aminohydrolase preparations have typical absorption spectra consistent with the absence of tightly bound prosthetic groups. No dissociable cofactors are necessary for catalysis since extensive dialysis of enzyme or addition of the metal chelator ethylenediaminete-traacetate (EDTA) did not cause significant inhibition. 

The idea that the same cofactor species operated in all Mo enzymes originated from a reconstitution assay. In this assay method, the isolated Moco from one enzyme, such as XO, is inserted into a cofactor-free mutant (Nit-1) of nitrate reductase from Neuraspora crassa, where it can reactivate or reconstitute normal nitrate reductase catalytic activity. It is now recognized that the Mo at the active site has many different coordination environments, as has been illustrated for the three Mo families in Fig. 1. In this context, the mutant nitrate reductase assay experiment is interpreted as involving some reprocessing of the inserted molybdenum cofactor from foreign enzymes to obtain the correct form of the cofactor for nitrate reductase catalysis. The Moco designation, if it is to be used, must refer to the family of sites present in Moco enzymes. 

G3P) and D-sedoheptulose 7-P as illustrated in Scheme 5.53. In addition D-erythrose 4-phosphate can function as the ketol acceptor thus producing D-fructose-6-P and G3P (Scheme 5.53). The enzyme relies on two cofactors for activity — thiamin pyrophosphate (TPP) and Mg2+—and utilizes the nucleophilic catalysis mechanism outlined in (Scheme 5.54).83 When TPP is used as a cofactor for nucleophilic catalysis, an activated aldehyde intermediate is formed. This intermediate functions as a nucleophile, and thus TK employs a strategy that is similar to the umpolung strategy exploited in synthetic organic chemistry. 

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