Enzymatic chain termination

Two basic protocols for DNA sequencing are commonly adapted in laboratories the chemical cleavage method (Maxam and Gilbert, 1977) and the enzymatic chain termination or dideoxy method (Sanger et al, 1977). Of the two approaches, the Sanger dideoxy... 

Enzymatic chain termination/dideoxy method (Smith, 1980)... 

Earlier in this chapter, it was mentioned that many of the nonprotein amino acids are components of nonribosomal peptides. During such a biosynthesis, the peptide is attached to a carrier protein through a thioester bond, until chain termination occurs and the final product is released. The carrier protein is posttranslationally modified by the attachment of a phosphopantetheinyl group from coenzyme A. This step gives rise to the active carrier protein with a phosphopantetheine arm upon which amino acids are added to during NRPS. As an example, loading of isoleucine onto the carrier protein is depicted below (Scheme 5). Further details about nonribosomal peptide syntheses and enzymatic reactions can be found in Chapter 5.19. 

Before the sequencing begins it is necessary to prepare a short primer that is complementary to a sequence at one end of the DNA strand to be sequenced. This may be prepared enzymatically,622 623 or by non-enzymatic synthesis. The short primer is annealed to the end of the DNA and the resulting molecule is incubated with a DNA polymerase and a mixture of the four mononucleotide triphosphates, one of which is radio-labeled in this position. Four reaction mixtures are prepared. Each mixture contains all four nucleoside triphosphates and also one of four different chain-terminating inhibitors, the most popular of which are the 2, 3 -dideoxyribonucleoside triphosphates ... 

The fatty acids in milk fat are derived from two sources, de novo synthesis of fatty acids in the mammary gland and plasma lipids (see Pal-quist, Chapter 2). De novo synthesis generally involves short-chain and medium-chain fatty acids and some 16 0. The proportions of various fatty acids depend on the specific balance between enzymatic chain elongation and chain termination. The plasma lipids are derived from the diet and also from storage in the body tissues. For non-ruminants, the diet has a large influence on the fatty acid composition but for ruminants, biohydrogenation in the rumen results in much less impact of diet on the final fatty acids absorbed into the bloodstream. 

Scheme 4.14 Enzymatic synthesis of end-functionalized poly(caprolactone) monomers using carboxylic acid as chain-terminating group.

The eukaryotic FASs synthesize predominantly the 16-carbon saturated product with smaller amounts of 14- and 18-carbon products. The enzymatic basis of product specificity of the animal FAS has been studied in some detail in G. Hammes and S. Smith s laboratories. The KS has a relatively broad chain-length specificity and is able to transfer efficiently saturated acyl moieties with 2-14 carbon atoms from the phosphopantetheine thiol to the active-site cysteine thiol. However, longer chain-length acyl moieties are transferred between thiols with increasing difficulty [17], In contrast, the chain-terminating thioesterase has very limited ability to remove acyl moieties with less than 16 carbon atoms from the phosphopantetheine thiol (S. Smith, 1978, 1991). Thus the specificities of... 

The first enzymatic step involves the covalent attachment of the first carbohydrate unit to the side chain of a specific amino acid residue. Further sugar residues are added in sequential order by specific glycosyltransferases for each of the sugar residues. It appears that specific multienzyme systems are required for the biosynthesis of each type of polymer (153). Frequently the carbohydrate chain terminates with an N-acetylneuraminic acid residue. 

In marked contrast to enzymatic degradation, the position of the ester group (chain end or within the chain) does not affect the hydrolytic degradability of PLA-based materials, as long as the polymers are composed of one monomer unit. An exception is the report on the acidic hydrolysis of amorphous poly(DL-lactide) (poly(DL-lactic acid) (PDLLA)) in the solid state by Shih [110,111], where the ester group adj acent to the chain terminal is more susceptible to hydrolytic degradation than that in the middle of the chain. Degradation at the chain end and in the middle of the chain is, respectively, called exo-... 

Lipases have also been used as initiators for the polymerization of lactones such as /3-bu tyro lac tone, <5-valerolactone, e-caprolactone, and macrolides.341,352-357 In this case, the key step is the reaction of lactone with die serine residue at the catalytically active site to form an acyl-enzyme hydroxy-terminated activated intermediate. This intermediate then reacts with the terminal hydroxyl group of a n-mer chain to produce an (n + i)-mer.325,355,358,359 Enzymatic lactone polymerization follows a conventional Michaelis-Menten enzymatic kinetics353 and presents a controlled character, without termination and chain transfer,355 although more or less controlled factors, such as water content of the enzyme, may affect polymerization rate and the nature of endgroups.360... 

Enzyme preparations from liver or microbial sources were reported to show rather high substrate specificity [76] for the natural phosphorylated acceptor d-(18) but, at much reduced reaction rates, offer a rather broad substrate tolerance for polar, short-chain aldehydes [77-79]. Simple aliphatic or aromatic aldehydes are not converted. Therefore, the aldolase from Escherichia coli has been mutated for improved acceptance of nonphosphorylated and enantiomeric substrates toward facilitated enzymatic syntheses ofboth d- and t-sugars [80,81]. High stereoselectivity of the wild-type enzyme has been utilized in the preparation of compounds (23) / (24) and in a two-step enzymatic synthesis of (22), the N-terminal amino acid portion of nikkomycin antibiotics (Figure 10.12) [82]. 

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