Enzyme amino acid incorporating

Incorporation of biologically important molecules into LDHs has become of interest in recent years and materials such as DNA [189], ATP [190], amino acids or enzymes [191,192] and vitamins [193] can be stabilized in the interlayer space of LDHs. If enzymes and proteins, for example, can be immobilized in the interlayer galleries of LDHs, new types of selective catalysts (see Sect. 2.2) as well as new delivery systems and carrier materials can be expected. 

Jhe synthesis of proteins, as characterized by the in vitro incorporation of amino acids into the protein component of cytoplasmic ribonu-cleoprotein, is known to require the nonparticulate portion of the cytoplasm, ATP (adenosine triphosphate) and GTP (guanosine triphosphate) (15, 23). The initial reactions involve the carboxyl activation of amino acids in the presence of amino acid-activating enzymes (aminoacyl sRNA synthetases) and ATP, to form enzyme-bound aminoacyl adenylates and the enzymatic transfer of the aminoacyl moiety from aminoacyl adenylates to soluble ribonucleic acid (sRNA) which results in the formation of specific RNA-amino acid complexes—see, for example, reviews by Hoagland (12) and Berg (1). The subsequent steps in pro-... 

In most peptides synthetases an epimerization domain that mediates D-amino acid incorporation is embedded within the module. Thus, L-amino acids are substrates of the synthetase and in situ epimerization occurs during peptide chain elongation. The first module of gramicidin S synthetase from Bacillus brevis is a well studied example [39]. This module recognizes L-phenylalanine, and the tightly bound intermediate is epimerized to fhe D-Phe-enzyme com-... 

Although the accuracy of translation (approximately one error per 104 amino acids incorporated) is lower than those of DNA replication and transcription, it is remarkably higher than one would expect of such a complex process. The principal reasons for the accuracy with which amino acids are incorporated into polypeptides include codon-anticodon base pairing and the mechanism by which amino acids are attached to their cognate tRNAs. The attachment of amino acids to tRNAs, considered the first step in protein synthesis, is catalyzed by a group of enzymes called the aminoacyl-tRNA synthetases. The precision with which these enzymes esterify each specific amino acid to the correct tRNA is now believed to be so important for accurate translation that their functioning has been referred to collectively as the second genetic code. 

As previonsly indicated, the digestion of proteins is ordinarily considered to occur successively via the gastric, pancreatic, and intestinal stages, as per the respective sources of the digestive enzymes. Gastric juices include proteases of the pepsin family, as well as hydrochloric acid. Pancreatic juice contains a number of protease enzymes snch as trypsin, chymotrypsin, and elastase, and some are noted to involve a serine residne, serine being 1 of the 20 amino acids incorporated into protein molecules. Hence, the term sometimes nsed is serine protease. 

All the amino acids incorporated into proteins in the body are stable under physiological conditions. However, some are easily modified by chemical or enzymatic reactions while others are difficult to change. The more reactive amino acids are those with acidic or basic groups on the side-chain (aspartate, glutamate, arginine, histidine, lysine) or sulfhydryl (Fig. 3.2) or hydroxyl groups (cysteine, serine, threonine). The more reactive amino acids are often part of enzyme active sites (Chapter 7) where they participate in the reaction catalyzed by the enzyme. 

The concept of enzyme subsites for binding of the several amino acid residues on either side of the scissile bond to the enzyme was introduced first by Schechter and Berger (27, 28) from their studies on the substrate specificity of papain. This concept of subsites will have special relevance to the specificity of amino acid incorporation during the resynthesis reaction to be discussed later. 

Aso et al. [95] studied a model system in order to obtain basic information on the mechanism of amino acid incorporation during an enzymatic modification reaction in the presence of papain. They found that the amino acid ester reacted as a nucleophile in the aminolysis of the acyl-enzyme intermediate to result in the formation of new peptides. Several proteases used in enzymatic peptide bond synthesis are known to form transitory acyl-enzyme intermediates during the hydrolysis of proteins. However, the acyl groups can be transferred to other nucleophiles (amino terminals of peptides or amino acids), synthesizing new peptide bonds [71]. With full knowledge of the above-mentioned facts, covalent amino acid enrichment of proteins can result in... 

Methionine-enriched protein was produced also from an enzymatically prehydrolyzed milk protein using an enzymatic peptide modification method with a-chymotrypsin as catalyst. Amino acid incorporation leading to methionine enrichment of the product proceeded via formation of covalent bonds. The concentration of the substrate was 25% (w/v). Methionine was added to the reaction mixture in the form of methionine methyl ester hydrochloride. An ester/substrate ratio of 1 5 was used in the enzymatic peptide modification reaction. The methionine content of the product was twice as high as that of the substrate. The slight change in the degree of hydrolysis revealed that part of the amino acids were bound to the peptide chains and that transpeptidation was the main force during this enzyme-catalyzed reaction. The newly incorporated Met was located in C- and N-termi-nals in a ratio of 3 1 [82],... 

Recent studies also supported covalent amino acid incorporation during the enzyme-catalyzed modification reaction [105,106], and proteolysis in organic solvents is also mentioned as a particular way of amino acid incorporation involved in aminolysis [107]. 

Feedback repression is the inhibition of formation of one or more enzymes in a pathway by a derivative of the end product. In many (but not all) amino acid biosynthetic pathways, the amino add end product must first combine with its transfer RNA (tRNA) before it can cause repression. Feedback repression is a widespread regulatory device especially for the synthesis of molecules intended for incorporation into macromolecules, e.g. amino adds, purines, and pyrimidines. Synthesis of vitamins also appears to be controlled by feedback repression, as well as by catabolite regulation (Birnbaum et al, 1967 Sasaki, 1965 Newell and Tucker, 1966 Wilson and Pardee, 1962 Papiska and Lichstein, 1968). Regulation of vitamin synthesis is important since only a small number (probably about 1000) of vitamin molecules are required per cell whereas many molecules of an average amino acid (probably 50 million) are required. An extremely wasteful case of vitamin overproduction would develop if enzymes for vitamin synthesis were produced at the same rate and were as active as the amino acid biosynthetic enzymes. 

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