Lysine chemical modification reagents

Chemical modification reagents for labelling reactive amino acid side chains (predominantly cysteine or lysine) have been available for many years and used to probe for residues dose to or in the active site of enzymes. Although fluorescent derivative,s of these reagents have been used less frequently, they occasionally reward persistent experimentation by offering more detailed... 

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). 

An interesting observation is that an enzyme may exhibit different pH activity profiles for various neutral substrates. The explanation of this is that the enzyme binds or transforms such various substrates differently. For example. Taka amylase has different pH optima for long chain amyloses and for low molecular mass substrates. Some specific chemical modifications of the side chains of the enzyme may also alter the pH activity profiles. Kobayashi, Miura and Ichisima (1992) modified the lysine amino groups using bifimctional reagent o-phtalaldehyde, and observed a pronounced shift in the pH-dependence of ohgomaltoside hydrolysis. 

Analyses for lysine after acid hydrolysis of proteins are usually quantitative and reliable, unless the lysines have been exposed to certain types of reagents (e.g. cyanate, nitrous acid). In a few proteins some lysines are fi-N-methylated (see 2.12.1.2) the methyllysines are stable to acid hydrolysis and emerge from many analyzer columns as a shoulder on the trailing edge of lysine, thus making lysine quantitation less precise (see 2.12.1). Methyllysine may also be formed in proteins by chemical modification ( 3.1.1.3). Many derivatives of lysine are not stable to acid hydrolysis (e.g. s-N-acetyllysine) and will not be discussed here, but those which occur naturally in proteins are discussed in 2.12,1. 

The unexpected specificity which can be achieved with functional group modification reagents is an apparent consequence of the native protein s ability to impose a unique chemical environment on a given amino acid under a given set of experimental conditions. It is important to emphasize that the site-specific modification of a protein is a kinetic phenomenon and selective modifications result from the ability of the protein to alter the reaction rate of a single residue under one clearly defined condition of pH, ionic strength and temperature. For example, it is entirely possible that if, at pH 7.0, one lysine residue is substantially more reactive than either free lysine or other lysine residues in the protein it may well be less reactive than these at pH 9.0. 

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. 

We have designed and/or studied several families of mono- and polynuclear organometallic complexes aimed at the selective and covalent chemical modification of proteins through their lysine residues. Chronologically, the first reagents included an N-hydroxysuccinimide ester function that is known from classical... 

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