Chemical modification of amino acid residues

Many Proteins Undergo Chemical Modification of Amino Acid Residues... 

Although the AalT molecule has several characteristics in common with other toxins isolated from A. australis, such as a conserved hydrophobic surface conducive to binding to its receptor, AalT is unique in that it possesses an atypical disulfide bridge and an unusually long C-terminus (42). It has also been demonstrated that chemical modification of amino acid residues of the AalT molecule results in a decreased toxicity level to insects (47). 

From studies of chemical modification of amino acid residues studied by many investigators, it was shown that the ones located in loop B are essential for neurotoxicity. For instance, the Arg-31, Arg-34, Trp-37, Tyr-23, Lys-24, and Lys-25 are known to be related to neurotoxicity. It is logical to assume that loop B is most likely to bind to the AChR. 

The behavior of immobilized enzymes differs from that of dissolved enzymes because of the effects of the support material, or matrix, as well as conformational changes in the enzyme that result from interactions with the support and covalent modification of amino acid residues. Properties observed to change significantly upon immobilization include specific activity, pH optimum, Km, selectivity, and stability.23 Physical immobilization methods, especially entrapment and encapsulation, yield less dramatic changes in an enzyme s catalytic behavior than chemical immobilization methods or adsorption. The reason is that entrapment and encapsulation result in the enzyme remaining essentially in its native conformation, in a hydrophilic environment, with no covalent modification. 

Attempts have been made to study the active site by chemical modification of amino acid side chains (Messner e/ aL, 1970 Thrasher / a/., 1975 Thrasher and Cohen, 1971). No attempts have been made to separate the various products of the modification reactions and to study the individual homogeneous populations of modified proteins. Collectively, however, the results of these studies would appear to implicate an amino group in the cytophilic site. The data of Ciccimarra et al. (1975) suggest that there are two lysine residues in a decapeptide containing the site, but the positions of the modified amino groups have not been ascertained, nor has the effect of these reagents on other side chains and on conformations been studied. 

Chemical modification of amino acid side chain functionalities will also serve to cleave specific peptide bonds selectively. Chemical cleavage of a polypeptide chain exploits the unique reactivity of chemically modified side chains of particular amino acids in the labilization of adjacent peptide bonds by neighbouring group participation (68). The residues investigated so far for this purpose have been methionine, cysteine and the aromatic amino acids including tryptophan (438-440, 443). 

Other PTMs may involve changes in the chemical nature of amino acids (e.g., citrullination or deimination). Because many of these modifications result in mass changes that are measurable by MS, they are amenable to detection by MS-based approaches. A number of emerging MS-based strategies allow the identification of PTMs. Several MS-based methods to determine the types and sites of protein phosphorylation and ubiquitination have been developed. Phosphorylation occurs mainly on serine, threonine, and tyrosine residues at a frequency ratio of 1800 200 1 in vertebrates.70 Although the phosphorylation of tyrosine residues occurs less frequently in the proteome, it has been extensively studied. 

Another major effect, found in PGA, is optical inversion of L-glutamate to D-glutamate residues. One implication of the radiation-induced optical inversion in proteins is that some modification of amino acids may pass undetected by the usual chemical analyses which do not distinguish between l- and D-isomers. Furthermore, introducing a D-amino acid residue into a protein could have a far-reaching effect on the secondary and tertiary structures, and this could have a more serious effect on the functional properties of the molecule than changes in the side chains. One biological property of PGA which is affected by irradiation in solution is its hydrolysis by proteolytic enzymes. The conformation of the polymer has a marked effect on its susceptibility to hydrolysis by certain enzymes 27), and we have... 

Ideally, chemical modification should be specific for only one type of amino acid residue, and should have minimal effects on the structure and function of the protein. These ideals are met only rarely, and consequently there are limitations to the conclusions that can be drawn from chemical modification experiments. Table 5-6 lists several reagents in common use. Some of the most popular of these, which are used in chemical modification of residues in the active sites of enzymes, are considered more fully below. 

Subsequent to their synthesis, most proteins are modified by the addition of various chemical groups to amino acid residues. These modifications, which alter protein structure and function. Include acetylation, hydroxylatlon, glycosylatlon, and phosphorylation. 

If we look at the experimental and theoretical aspects of heterogeneous catalysis, we will see that the state of affairs here is even worse than in enzyme catalysis. This is mainly due to the fact that it is easier to perform a reproducible experiment with purified enzymes than in the case of many solid catalysts. To be sure, enzymes are complicated substances, and it is necessary to be extremely careful during their preparatory isolation. However, being synthesized within the cell of a matrix, enzyme molecules represent practically identical copies. Therefore, the reproducibility of their functioning is, as a rule, much better than that of the solid catalysts of abiogenic origin. The identity of enzyme molecules is limited, however, by the possibility of the existence of plural forms of the enzymes, that are determined, in turn, by the chemical composition and the primary chemical structure (the sequence of amino acids residues in the polypeptide chain) of a protein molecule. Most proteins are sufficiently complex, and thus can exist in several conformational modifications. 

In the bovine heart mitochondrial ADP/ATP carrier, there are four cysteine residues Cys , Cys , Cys and Cys which are all at intervals of about 100 amino acid residues except Cys . This paper deals with the molecular mechanism of substrate transport by the carrier, deduced mainly from the effects of chemical modifications of its cysteine residues by SH-reagents under various conditions. The important role of the loops of the carrier in its transport function is described. Unless otherwise noted, the results were obtained with bovine heart submito-chondrial particles, in which the orientation of membrane proteins is inside-out relative to that of mitochondria. 

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