Enzyme Catalyzed Bond Formation

In the absence of acids or bases peptide bonds are quite resistant to hydrolysis, but their hydrolytic cleavage is extremely accelerated in the presence of proteolytic enzymes. The remarkable catalytic effect of these enzymes tempted many investigators, through a long period of time (Fruton 1982), to adopt them for synthesis, rather than hydrolysis of peptide bonds. Since enzymes are catalysts and merely accelerate the establishment of equilibria, it is possible to use proteolytic enzymes for amide bond formation if the equilibrium of the reaction can be modified. Thus anilides of blocked amino acids could be prepared with the help of papain  

Aniline was used in large excess and the anilide, being insoluble in water separates from the reaction mixture. Both factors shift the equilibrium to the right and therefore the anilide could be obtained in high yield. A practical application of this approach is the resolution of an enantiomeric mixture only the L-derivative (of an N-acyl-amino acid) is converted, the D-amino acid derivative remains unchanged and can easily be separated from the insoluble anilide of the L-compound. 

A more general use of proteolytic enzymes in peptide synthesis became feasible with the discovery (Sealock and Laskowsky 1969) of the effect of water miscible organic solvents on the equilibrium in enzyme catalyzed peptide bond hydrolysis and synthesis. In the presence of isopropanol (or dimethylfor-mamide, etc.) the dissociation of the carboxyl group is suppressed and, at least in a selected pH region, the equilibrium is shifted toward synthesis. A notable case is the conversion of porcine insulin to human insulin. Enzymatic cleavage of the C-terminal residue of the B-chain (alanine) with carboxypeptidase yields desalanino pork insulin. This cleavage is followed by the incorporation of 

L-threonine, in the form of its tertiary butyl ester, used in large excess and in the presence of isopropanol  

Acidolytic removal of the tertiary butyl group completes the conversion. This transformation is being carried out on a commercial scale. 

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S. Except for oxido-reductases, transferases, and hydrolases, most ligases (enzymes that catalyze bond formation) are entirely substrate specific. Thus, fumarate hydratase (or fumarase) reversibly and stereospecifically adds water to fumaric acid to produce (S)-( — )-malic acid only (8) (Figure 1), and another enzyme, mesaconase, adds water to mesaconic acid to form (+ )-citramalic acid (9) (Figure 2). Although no extensive studies are available, it appears that neither fumarase nor mesaconase will add water stereospecifically to any other a,(3-unsaturated acid. 

Other ThDP enzymes catalyze the formation of C-C bonds by transferring C2 units from a donor compound to an acceptor compound (transferase activity). We have studied the E. coli transketolase with respect to structure-function relationships, as well as to possible applications in asymmetric syntheses (Section 2.2.2.2.1). During the late 1990s a transketolase-like enzyme, 1-deoxyxylulose 5-phosphate synthase, was discovered. Its structure and value in chemoenzymatic syntheses were also assessed in project B21 (Section 2.2.2.2.2). 

The joining of Okazaki fragments requires an enzyme that catalyzes the joining of the ends of two DNA chains. The existence of circular DNA molecules also points to the existence of such an enzyme. In 1967, scientists in several laboratories simultaneously discovered DNA ligase. This enzyme catalyzes the formation of a phosphodiester bond... 

A specific type of lyase, the aldolase class of enzymes, catalyzes the formation of an asymmetric C-C bond, which is a most useful reaction to the synthetic... 

Mechanistic aspects of P-0 bond formations and cleavages have been reviewed 15 and are outside the scope of this work. The use of enzymes catalyzing the formation... 

Enzymes catalyze the formation of carbon-carbon bonds between allylic and homoallylic pyrophosphate species by mechanisms that are very different from those for carbonyl compounds. Here, carbonium ions, stabilized as ion pairs and generated from allylic pyrophosphates, are likely to be the intermediates that add to the TT-electron density of carbon-carbon double bonds to form new carbon-carbon single bonds. Reaction patterns are consistent with model systems and the mechanisms are based on analogies with the models, stereochemical information (which is subject to interpretation), and the structural requirements for inhibitors. Detailed kinetic studies, including isotope effects, which provide probes in the aldolase and Claisen enzymes discussed in Section II, have not yet been performed in these systems. The possibility for surprising discoveries remains and further work is needed to confirm the proposed mechanisms and to generalize them. 

This enzyme catalyzes the formation of phosphoester bonds between the two pieces of DNA. 

Linkage specificity—an enzyme catalyzes the formation or breakage of only one type of bond. 

The individual modules of polyketide synthases (PKSs) and of NRPSs represent another example of modularity. An electrophile and a nucleophile are covalently attached to two different subunits of a multidomain enzyme. The selectivity for the electrophile resides in the domain catalyzing bond formation between two substrates and the selectivity for the nucleophile is controlled by a transfer domain which attaches the nucleophile onto a carrier domain. ... 

Our model study (23-25) is believed to be the first rigorous study to demonstrate biocatalysis at silicon. This data suggests that homologous lipase and protease enzymes catalyze the formation of siloxane bonds under mild conditions. 

Hinderberger, D, Piskorski RP, Goenrich G, Thauer RK, Schweiger A, Harmer J, Jaim B. 2006. A nickel-alkyl bond in an inactivated state of the enzyme catalyzing methane formation, Chem, 45 3602-3607. 

The hydrolases are enzymes that break bonds with the incorporation of water and that, under certain conditions, can catalyze bond formation. A large number of hydrolases are needed to degrade starch to glucose. They belong to the subgroup of the glycosidases. 

The enzyme catalyzed reactions that lead to geraniol and farnesol (as their pyrophosphate esters) are mechanistically related to the acid catalyzed dimerization of alkenes discussed m Section 6 21 The reaction of an allylic pyrophosphate or a carbo cation with a source of rr electrons is a recurring theme m terpene biosynthesis and is invoked to explain the origin of more complicated structural types Consider for exam pie the formation of cyclic monoterpenes Neryl pyrophosphate formed by an enzyme catalyzed isomerization of the E double bond m geranyl pyrophosphate has the proper geometry to form a six membered ring via intramolecular attack of the double bond on the allylic pyrophosphate unit... 

Cyanohydrin Synthesis. Another synthetically useful enzyme that catalyzes carbon—carbon bond formation is oxynitnlase (EC 4.1.2.10). This enzyme catalyzes the addition of cyanides to various aldehydes that may come either in the form of hydrogen cyanide or acetone cyanohydrin (152—158) (Fig. 7). The reaction constitutes a convenient route for the preparation of a-hydroxy acids and P-amino alcohols. Acetone cyanohydrin [75-86-5] can also be used as the cyanide carrier, and is considered to be superior since it does not involve hazardous gaseous HCN and also virtually eliminates the spontaneous nonenzymatic reaction. (R)-oxynitrilase accepts aromatic (97a,b), straight- (97c,e), and branched-chain aUphatic aldehydes, converting them to (R)-cyanohydrins in very good yields and high enantiomeric purity (Table 10). 

Figure 6.8 Schematic diagram of the enzyme DsbA which catalyzes disulfide bond formation and rearrangement. The enzyme is folded into two domains, one domain comprising five a helices (green) and a second domain which has a structure similar to the disulfide-containing redox protein thioredoxin (violet). The N-terminal extension (blue) is not present in thioredoxin. (Adapted from J.L. Martin et al.. Nature 365 464-468, 1993.)...

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