Covalent attachment of amino acids

Increases in nutritional quality have been achieved through the covalent attachment of amino acids by chemical or enzymatic modifications. 

Supplementation Through Covalent Attachment of Amino Acids... 

The chemical methods already available for the covalent attachment of amino acids to proteins include those used to modify carboxyl and amino groups of proteins. 

Figure 2. General scheme for covalent attachment of amino acids to proteins by the active-esters method. Reaction conditions are described in Ref. 22 TFA = trifluoroacetic acid.

Figure 3. Structure of peptide and isopeptide bonds resulting from covalent attachment of amino acids to proteins by chemical methods. In isopeptide bond formation Rt = -CH2- or -CH2CH2- of aspartic or glutamic acid and R2 = -(CH2)n- of lysine.

Some physical and functional properties of casein modified by the covalent attachment of amino acids are given in Table IX. Despite extensive modification, the relative viscosities of 2% solutions of the modified proteins did not change significantly, with the exception of aspartyl casein which was more viscous. There was some decrease in the solubilities of aspartyl casein and tryptophyl casein as compared with the casein control. It is anticipated that adding some 11.4 tryptophyl residues per mole of casein would decrease the aqueous solubility of the modified protein. However the results with aspartyl casein are unexpected. The changes in viscosity, solubility, and fluorescence indicate that aspartyl casein is likely to be a more extended molecule than the casein control. There was a marked decrease in the fluorescence of aspartyl casein and tryptophyl casein (see Table IX). The ratios of the fluorescences of acetylmethionyl casein to methionyl casein and t-BOC-tryptophyl casein to tryptophan casein were 1.20 and 2.01, respectively, indicating the major effects that these acyl groups have on the structure of the casein. 

In 1975, the fabrication of a chiral electrode by permanent attachment of amino acid residues to pendant groups on a graphite surface was reported At the same time, stimulated by the development of bonded phases on silica and aluminia surfaces the first example of derivatized metal surfaces for use as chemically modified electrodes was presented. A silanization technique was used for covalently binding redox species to hydroxy groups of SnOj or Pt surfaces. Before that time, some successful attemps to create electrode surfaces with deliberate chemical properties made use of specific adsorption techniques... 

In this part we will describe recent achievements in the development of biosensors based on DNA/RNA aptamers. These biosensors are usually prepared by immobilization of aptamer onto a solid support by various methods using chemisorption (aptamer is modified by thiol group) or by avidin-biotin technology (aptamer is modified by biotin) or by covalent attachment of amino group-labeled aptamer to a surface of self-assembly monolayer of 11-mercaptoundecanoic acid (11-MUA). Apart from the method of aptamer immobilization, the biosensors differ in the signal generation. To date, most extensively studied were the biosensors based on optical methods (fluorescence, SPR) and acoustic sensors based mostly on thickness shear mode (TSM) method. However, recently several investigators reported electrochemical sensors based on enzyme-labeled aptamers, electrochemical indicators and impedance spectroscopy methods of detection. 

Plastein Formation. The ability of some proteolytic enzymes to convert soluble proteins to an insoluble aggregate was noted as early as 1947 ( 42,43) in the formation of plakalbumin from albumin by subtilisin. More recently, Fujimaki and co-workers have investigated this reaction for removal of flavor constituents from soybeans as well as a method of covalently attaching essential amino acids to the protein. 

Any chemist knows that the more steps there are in a chemical synthesis, the lower the final yield. For example, if each step in a 10-step synthesis furnishes a 90 percent yield of product, the yield of the final product will be only about 35 percent. That is why it is not possible to extend Du Vigneaud s masterful syntheses (see chapter 6) of the hormones oxytocin and vasopressin (9 amino acid residues each) to proteins, even small ones such as ribonuclease A (124 amino acid residues). In order to pursue this daunting challenge Robert Bruce Merrifield (1921-2006), at Rockefeller University, devised a new concept solid-phase synthesis. The idea is disarmingly simple covalently attach an amino acid to a macroscopic particle that can be exposed to the reaction, washed, and then separated by simple filtration. Each reaction step requires no chromatography and no crystallization, just washing and filtering. At the end, completed peptide chains are chemically released from the particles. 

GA4-17-S-propyl-S-phenacyl azide (structure I, Fig. 3) induces a-amylase in aleurone protoplasts (Fig. 1), although it is less active than the parent compound (GA4). In aqueous solution it has a X ax of294 nm (Fig. 2). Solutions are stable in the dark but are photolyzed within seconds of exposure to intense light of wavelengths above 300 nm (Fig. 2). Aryl azides photolyse to highly reactive aryl nitrenes that can covalently attach to amino acids at the binding site. See [15] for a detailed. account of the reactions of aryl nitrenes. 

VII. COVALENT ATTACHMENT OF FATTY ACIDS TO AMINO ACID RESIDUES IN PROTEINS... 

FIG. 9 Covalent attachment of fatty acid to L-methionine catalyzed amino acylase... 

Evolution has provided the cell with a repertoire of 20 amino acids to build proteins. The diversity of amino acid side chain properties is enormous, yet many additional functional groups have been selectively chosen to be covalently attached to side chains and this further increases the unique properties of proteins. Diese additional groups play a regulatory role allowing the cell to respond to changing cellular conditions and events. Known covalent modifications of proteins now include phosphorylation, methylation, acetylation, ubi-quitylation, hydroxylation, uridylylation and glycosyl-ation, among many others. Intense study in this field has shown the addition of a phosphate moiety to a protein... 

Fig. 8.2 Model for the synthesis of amino acids from alpha-keto acid precursors covalently attached to dinucleotides. The dinucleotide that is capable of catalyzing synthesis of particular amino acids is proposed to contain the first two bases of the codon specifying that amino acid (Copley et al., 2005)...

The novel concept of synthesizing a molecule while attached to a swollen cross-linked resin bead was introduced and demonstrated by R. B. Merrifield with the solid-phase peptide synthesis method about 20 years ago (1,2). The procedure involves the covalent attachment of an amino-acid residue to the polymer bead followed by the addition of subsequent amino-acid units in a stepwise manner under conditions that do not disrupt the attachment to the support. At the completion of the assembly of the peptide, the product is cleaved from the resin and recovered. The macro-scopically insoluble support provides convenient containment of the desired product so that isolation and purification from soluble co-products in the synthesis can be achieved by simple... 

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]. 

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