Amino acid residues, chemical modification

Asp-49/35 is essential to optimal calcium binding and catalysis. Replacement of Asp-49 with other amino acids or chemical modification of the side-chain carboxylate reduces Acat to less than 5% of native rates (Fleer etai, 1981 Van den Berghe/a/., 1989). The close spatial coupling of Asp-49/35 with its respective catalytic histidine (His-48/34) ensures a fixed active site geometry. Stability of this bihelical substructure is critical because His-48/34 is supported by a side chain from an adjacent segment of the same helix (Tyr-52) and must hydrogen bond with a residue from the opposed helix (Asp-99/64) for function. 

Kievan and Tse (1983) have examined the effect of chemical modification of E. coli topoisomerase I and DNA gyrase by tetranitromethane, which reacts preferentially with tyrosine residues. With each enzyme, treatment with tetranitromethane led to abolition of the topoisomerase activity. Moreover, the enzymes were protected from this inactivation when bound to DNA, implying that some of the modified residues are involved in DNA binding. However, this study does not identify the tyrosine residues involved in the protein-DNA bond as being the amino acid residues whose modification inactivates the enzyme. In the case of DNA gyrase, which has two subunits, it was not determined which subunit was inactivated. 

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. 

Neurotoxins present in sea snake venoms are summarized. All sea snake venoms are extremely toxic, with low LD5Q values. Most sea snake neurotoxins consist of only 60-62 amino acid residues with 4 disulOde bonds, while some consist of 70 amino acids with 5 disulfide bonds. The origin of toxicity is due to the attachment of 2 neurotoxin molecules to 2 a subunits of an acetylcholine receptor that is composed of a2 6 subunits. The complete structure of several of the sea snake neurotoxins have been worked out. Through chemical modification studies the invariant tryptophan and tyrosine residues of post-synaptic neurotoxins were shown to be of a critical nature to the toxicity function of the molecule. Lysine and arginine are also believed to be important. Other marine vertebrate venoms are not well known. 

Chemical modifications of proteins (enzymes) by reacting them with iV-acylimidazoles are a way of studying active sites. By this means the amino acid residues (e.g., tyrosine, lysine, histidine) essential for catalytic activity are established on the basis of acylation with the azolides and deacylation with other appropriate reagents (e.g., hydroxylamine). 

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... 

From kinetic (57) and chemical modification (58) studies, Hurst et al. concluded that the catalytic residues in a cellulase from Aspergillus niger are a carboxylate anion (pKa 4.0-4.5) and a protonated carboxyl group (pKa 5.0-5.5) with tryptophan and dicarboxylic amino acid residues involved in substrate binding. 

Oxidation of two out of 13 tryptophan residues in a cellulase from Penicillium notatum resulted in a complete loss of enzymic activity (59). There was an interaction between cellobiose and tryptophan residues in the enzyme. Participation of histidine residues is also suspected in the catalytic mechanism since diazonium-l-H-tetrazole inactivated the enzyme. A xylanase from Trametes hirsuta was inactivated by N-bromosuc-cinimide and partially inactivated by N-acetylimidazole (60), indicating the possible involvement of tryptophan and tyrosine residues in the active site. As with many chemical modification experiments, it is not possible to state definitively that certain residues are involved in the active site since inactivation might be caused by conformational changes in the enzyme molecule produced by the change in properties of residues distant from the active site. However, from a summary of the available evidence it appears that, for many / -(l- 4) glycoside hydrolases, acidic and aromatic amino acid residues are involved in the catalytic site, probably at the active and binding sites, respectively. 

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