Lysine side-chain acylation

Since lysinoalanine and at least one D-amino acid are toxic to some animals (35), we wished to distinguish their effects in alkali-treated proteins. Such discrimination is possible, in principle, since we have found that acylating the e-amino group of lysine proteins seems to prevent lysinoalanine formation. Since lysinoalanine formation from lysine requires participation of the e-amino group of lysine side chains, acylation of the amino group with acetic anhydride is expected to prevent lysinoalanine formation under alkaline conditions if the protective effect survives the treatment. This is indeed the case (16). 

Protein functional groups able to react with anhydrides include the oc-amines at the N-terminals, the s-amine of lysine side chains, cysteine sulfhydryl groups, the phenolate ion of tyrosine residues, and the imidazolyl ring of histidines. However, acylation of cysteine, tyrosine, and histidine side chains forms unstable complexes that are easily reversible to regenerate the original group. Only amine functionalities of proteins are stable to acylation with anhydride reagents (Fraenkel-Conrat, 1959 Smyth, 1967). 

The intermediate can, however, also be trapped by an amine to form an amide although at pH 7 in aqueous solution primary amines are predominantly proto-nated and only poorly reactive. Intramolecularity will, however, improve the poor reactivity of a lysine residue towards an acyl intermediate provided that the His and the Lys residues are close in space. The net reaction under these conditions is therefore an amidation of the lysine side chain by the active ester that is more efficient than the direct acylation of a lysine residue by at least three orders of magnitude (Fig. 10). The lysine residue will also improve the reactivity of the His side chain by electrostatic transition state stabilization and the wasteful reaction with other His residues that gives rise to hydrolysis is therefore suppressed. 

F. 20.10. Function of lipoate. Lipoate is attached to the -amino group on the lysine side chain of the tranacylase enzyme (E2). The oxidized liptoate disulfide form is reduced as it accepts the acyl group from thiamine pyrophosphate (TPP) attached to Ej. The example shown is for the a-ketoglutarate dehydrogenase complex. 

As mentioned before, N-hydroxysuccinimide esters readily acylate the primary amine groups of proteins (that is the lysine side chain and the N-terminal residue). High acylation yields are obtained at basic pH, providing aminolysis of the N-hydroxysuccinimide ester is faster than its hydrolysis (Scheme 6.13). 

Effect of acylation. Formation of lysinoalanine from lysine re-quires the participation of an e-amino group of a lysine side chain. Therefore, it was expected that protection of amino groups by acylation (acetylation, succinylation, etc.), would reduce ly-sinoalanine formation under alkaline conditions, if the protective group (s) survived the treatment. Results in Tables strikingly demonstrate that acetylation of wheat gluten prevents lysinoala-nine formation. 

The amine containing side chains in lysine, arginine, and histidine typically are exposed on the surface of proteins and can be derivatized with ease. The most important reactions that can occur with these residues are alkylation and acylation (Figure 1.8). In alkylation, an active... 

The side-chain amino group of lysine is a strong nucleophile, the reactivity of which cannot be suppressed by protonation, so it must be protected at all times. Acyl groups such as formyl, which is stable to alkali, ammonia, and hydrogenation but sensitive to mild acid, and trifluoroacetyl (see Section 3.9), which is stable to piperidine and... 

Long-acting the threonine is deleted at B30 and the lysine at B28 is acylated with a miristoyl side-chain (B29Lys (e-tetradecanoyl)desB30)... 

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