Amino acid enzyme-catalyzed attachment

Transfer RNA precursors may undergo further posttranscriptional processing. The 3 -terminal trinucleotide CCA(3 ) to which an amino acid will be attached during protein synthesis (Chapter 27) is absent from some bacterial and all eukaryotic tRNA precursors and is added during processing (Fig. 26-23). This addition is carried out by tRNA nucleotidyltransferase, an unusual enzyme that binds the three ribonucleoside triphosphate precursors in separate active sites and catalyzes formation of the phosphodiester bonds to produce the CCA(3 ) sequence. The creation of this defined sequence of nucleotides is therefore not dependent on a DNA or RNA template—the template is the binding site of the enzyme. 

Enzyme-catalyzed attachment of amino acids to proteins represents an attractive and interesting way for improving the nutritional value of food proteins. The enzymes that participate in the gastrointestinal digestion of food proteins catalyze exclusively hydrolytic reactions under physiological conditions. However the synthetic activity of proteolytic enzymes was reported first by Danilewski in 1886, and more recently a number of studies have been devoted to plastein formation from con-... 

There is a family of enzymes that catalyze the attachment of amino acids to then-cognate tRNAs, aminoacyl-tRNA synthetases. There is one or more of these enzymes for each of the 20 amino acids that occur commonly in proteins. Each of these enzymes recognizes (a) a specific amino acid and (b) its cognate tRNA. Imagine a soup of 20 amino acids and 20 tRNAs, one for each amino acid. For example, the aminoacyl-tRNA synthetase for, saline would specifically pick valine out of the soup and catalyze its attachment to the tRNA for valine, tRNA . Simply, we can write the product of the reaction as val-tRNA . This is a lovely example of the role of molecular recognition in a critical life process. 

FTase catalyzes the covalent attachment of a farnesyl moiety via a thioether Unkage to the proteins bearing a C-terminal amino acid sequence known as the CAAX motif (Fig. 2) [12,21]. The farnesyl moiety is derived from farnesyl pyrophosphate (FPP), a 15-carbon isoprenyl intermediate in the mevalonate pathway of cholesterol biosynthesis. The binding of FPP to the enzyme has relatively high affinity (K = 1-lOnM), and FPP binding must precede the binding of the peptide substrate for successful catalysis [22,23]. 

MECHANISM FIGURE 22-18 Tryptophan synthase reaction. This enzyme catalyzes a multistep reaction with several types of chemical rearrangements. An aldol cleavage produces indole and glyceraldehyde 3-phosphate this reaction does not require PLP. Dehydration of serine forms a PLP-aminoacrylate intermediate. In steps and this condenses with indole, and the product is hydrolyzed to release tryptophan. These PLP-facilitated transformations occur at the /3 carbon (C-3) of the amino acid, as opposed to the a-carbon reactions described in Figure 18-6. The /3 carbon of serine is attached to the indole ring system. Tryptophan Synthase Mechanism... 

The second key advance was made by Mahlon Hoagland and Zamecnik, when they found that amino acids were activated when incubated with ATP and the cytosolic fraction of liver cells. The amino acids became attached to a heat-stable soluble RNA of the type that had been discovered and characterized by Robert Holley and later called transfer RNA (tRNA), to form aminoacyl-tRNAs. The enzymes that catalyze this process are the aminoacyl-tRNA synthetases. 

Vitamin B6 is a collective term for pyridoxine, pyridoxal, and pyridox amine, all derivatives of pyridine. They differ only in the nature of the functional group attached to the ring (Figure 28.10). Pyridoxine occurs primarily in plants, whereas pyridoxal and pyridoxamine are found in foods obtained from animals. All three compounds can serve as precur sors of the biologically active coenzyme, pyridoxal phosphate. Pyridoxal phosphate functions as a coenzyme for a large number of enzymes, par ticularly those that catalyze reactions involving amino acids. 

The attachment of an amino acid to a tRNA is catalyzed by an enzyme called aminoacyl-tRNA synthetase. A separate aminoacyl-tRNA synthetase exists for every amino acid, making 20 synthetases in total. The synthesis reaction occurs in two steps. The first step is the reaction of an amino acid and ATP to form an aminoacyl-adenylate (also known as aminoacyl-AMP). 

Recently a simplified process was developed for incorporating l-methionine directly into soy proteins during the papain-catalyzed hydrolysis (21). The covalent attachment of the amino acid requires a very high concentration of protein and occurs through the formation of an acyl-enzyme intermediate and its subsequent aminolysis by the methionine ester added in the medium. From a practical point of view, the main advantage of enzymatic incorporation of amino acids into food proteins, in comparison with chemical methods, probably lies in the fact that racemic amino acid esters such as D,L-methionine ethyl ester can be used since just the L-form of the racemate is used by the stereospecific proteases. On the other hand, papain-catalyzed polymerization of L-methio-nine, which may occur at low protein concentration (39), will result in a loss of methionine because of the formation of insoluble polyamino acid chains greater than 7 units long. 

Opening of a base pair in tRNAGln accompanies complexation with its cognate glutaminyl-tRNA synthetase [715]. All the tRNAs have an L-shaped three-dimensional structure as shown in Fig. 20.8. At one end of the L is the anticodon triplet it codes for the amino acid for which the respective tRNA is specific. At the other end of the L is the amino acid acceptor stem with the free O H of the terminal invariant adenosine, A76 in tRNAoln. The amino acid is attached here through an ester linkage in a reaction which is catalyzed by enzymes called synthetases. In contrast to the uniform tRNA structure, synthetases vary in amino acid sequence, molecular weight and subunit composition. 

As an illustration, consider the assay to measure the activity of the tRNA synthetases. These enzymes catalyze the covalent attachment to tRNA of an amino acid, usually radioactive as follows ... 

The catalytic mechanisms and molecular recognition properties of peptide synthetases have been studied for several decades [169]. Nonribosomal peptides are assembled on a polyenzyme-protein template, first postulated by Lipmann [170]. The polyenzyme model was refined into the thiotemplate mechanism (Fig. 11) in which the amino acid substrates are covalently bound via thioester linkages to active site sulfhydryls of the enzyme and condensed via a processive mechanism involving a 4 -phosphopantetheine carrier [171-173].The presence of a covalently attached pantetheine cofactor was first established in a cell-free system that catalyzed enzymatic synthesis of the decapeptides gramicidin S and tyrocidine. As in the case of fatty acid synthesis, its role in binding and translocating the intermediate peptides was analyzed [174,175]. 

Both classes of enzymes catalyze the common aminoacylation reaction but via different mechanisms (1). Class I and Class II aaRSs bind ATP in an extended and bent conformation, respectively (Fig. 2). In addition, class I enzymes bind the tRNA acceptor stem from the minor groove side, which orients the 2 -hydroxyl group of the A76 ribose for attachment of the amino acid (Fig. 3). In contrast. Class II aaRSs aminoacylate the 3 -hydroxyl of the terminal adenosine, because the enzyme binds to tRNA via its major groove. Class II phenylalanyl-tRNA synthetase (PheRS), which charges amino acids onto the 2 -hydroxyl group of A76 of tRNA , is the only known exception to this rule. 

Several enzyme-catalyzed reactions involve the nucleophilic attack of an active site amino acid side chain on the substrate, which results in an immediate reaction that is attached covalently to the enzyme. This type of catalysis is known as nucleophic (or covalent) catalysis. 

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