Lysine acylation

Determination of the completeness of lysine acylation is performed by dinitrophenylation (Fraenkel-Conrat et al. 1955) of the succinylated protein, followed by acid hydrolysis, and amino acid analysis. Since fi-dinitrophenyllysine is stable to acid hydrolysis, the recovery of lysine reflects the -succinyllysine content of the protein. 

Determination of Optimal pH for SC-PEG Reactivity. To triethanolamine-borate buffer (0.3 M, 1 mL) at the appropriate pH, a stock solution of N,a-acetyl-lysine (NAL) in water (50 mM, 0.1 mL) was added followed by a stock solution of SC-activated mPEG-5000 in CH3CN (50 mM active acyl, 0.1 mL). The resultant solution was vortexed and incubated at 28 °C for 1 h. A mixture of the same components but leaving out SC-PEG was used as a control. The TNBS assay version of Snyder and Sobocinski (18) was used to determine the unreacted NAL. 

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). 

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... 

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 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... 

FIGURE 6.30 Approaches for the synthesis of monosubstituted trifunctional amino acids. (A) Monoesterification of dicarboxylic acids. (B) Aa-Alkoxycarbonylation of lysine through the e-benzylidene derivative [Bezas Zervas, 1963]. (C) SelectiveN -detritylation of ditrityl derivatives.138 (D) A- AI ko x y met hy 1 at 10 n of histidine by displacement of AP-substituents.137 Cbz-His(CH2OR)-OMe are obtained from Cbz-His(xAc)-OMe. = Acylating reagent. 

Similarly, chemical hydrolysis of a number of a-amino acyl prodrugs of metronidazole (8.100, R=H see Sect. 8.5.4) was compared to the serum-catalyzed reaction [135][136]. The amino acids used for esterification included alanine, glycine, isoleucine, leucine, lysine, phenylalanine, and valine. Under physiological conditions of pH and temperature, ty2 values for hydrolysis in human serum ranged from 4.5 min for the Phe ester to 96 h for the lie ester. A good linear relationship was established between the log of the rate constant of enzymatic hydrolysis and the log of the rate constant of HO-cata-... 

Cleavage of the oxirane C-0 bond produces a zwitterionic intermediate (Fig. 10.22), which that can undergo chloride shift (Pathway a) to 2,2-dich-loroacetyl chloride (10.90) followed by hydrolysis to 2,2-dichloroacetic acid (10.91). Furthermore, the zwitterionic intermediate reacts with H20 or H30+ (Pathway b) by pH-independent or a H30+-dependent hydrolysis, respectively. The pH-independent pathway only is shown in Fig. 10.22, Pathway b, but the mechanism of the H30+-dependent hydrolysis is comparable. Hydration and loss of Cl, thus, leads to glyoxylyl chloride (10.92), a reactive acyl chloride that is detoxified by H20 to glyoxylic acid (10.93), breaks down to formic acid and carbon monoxide, or reacts with lysine residues to form adducts with proteins and cytochrome P450 [157], There is also evidence for reaction with phosphatidylethanolamine in the membrane. 

H. Cai, F. P. Guengerich, Acylation of Protein Lysines by Trichloroethylene Oxide , Chem. Res. Toxicol. 2000,13, 327 - 335 H. Cai, F. P. Guengerich, Reaction of Trichloroethylene and Trichloroethylene Oxide with Cytochrome P450 Enzymes Inactivation and Sites of Modification , Chem. Res. Toxicol. 2001, 14, 451 - 458. 

A Streptomyces enzyme that catalyzes hydrolysis of capsaicin is described by Koreishi et The substrate is an A -vanillyl aliphatic amide, and the authors found that their enzyme also accepted A lauroyl amino acids as substrates. The enzyme was used successfully to catalyze the reaction in the opposite direction, driving the equilibrium toward synthesis by running it in buffer containing 78% glycerol. Yields of 5-40% were obtained for a wide range of natural L-amino acids. In the case of L-lysine the enzyme catalyzed acylation at both amino groups, with a clear preference for the e-NH2. 

Some enzymes are nonfunctional until posttranslationally modified. Examples of these enzymes include the acyl- and carboxyltransferases. While lipoate and phosphopantetheine are necessary for acyl transfer chemistry, tethered biotin is used in carboxyl transfer chemistry. Biotin and lipoate tethering occur under a similar mechanism the natural small molecule is activated with ATP to form biotinyl-AMP or lipoyl-AMP (Scheme 20). A lysine from the target protein then attacks the activated acid and transfers the group to the protein. The phosphopantetheine moiety is transferred using its own enzyme, the phosphopantetheinyltrans-ferase (PPTase). The PPTase uses a nucleophilic hydroxy-containing amino acid, serine, to attach the phosphopantetheinyl (Ppant) arm found in coenzyme A to convert the apo (inactive) carrier protein to its holo (active) form. The reaction is Mg -dependent. 

Histidine residues are efficient nucleophiles in aqueous solution at pH 7, much more so than lysines, and this is the basis for the site-selective functionalization of lysine residues in folded polypeptides and proteins [24, 25]. p-Nitrophenyl esters react with His residues in a two-step reaction to form an acyl intermediate under the release of p-nitrophenol followed by the reaction of the intermediate with the most potent nucleophile in solution to form the reaction product. In aqueous solution the reaction product is the carboxylic acid since the hydroxide ion is the most efficient nucleophile at pH 7. If there is an alcohol present the reaction product will be an ester and the overall reaction is a transesterification reaction. 

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. 

Aside from the Maillard reaction, other covalent modifications of amino acids and proteins are possible within the caries lesion, which merit future investigation. For example, certain oral microorganisms excrete y-glutamyl transferases. These enzymes catalyse the formation of cross-links between glutamic acid and lysine residues of proteins. In addition, N-acyl amino acids are present in plaque, which adsorb to mineral surfaces. 

From Nocardia strains several closely related compounds (nocobactins, formo-bactin, amamistatins) were isolated that contain three typically Fe " binding sites, two hydroxamate units, and ahydroxyphenyloxazole stmcture (cf. Sect. 3.2 below). The C-terminus is A-hydroxy-cyc/o-Lys bound to a long chain 3-hydroxy fatty acid, whose hydroxy group is esterified by A -acyl-A -hydroxy-Lys, the a-amino group of which is bound to 2-o-hydroxyphenyl-5-methyl-oxazole-4-carboxylic acid (Table 4). For the amamistatins the configuration of the cyclic lysine was determined as L, the open one as d, and that of C-3 of the fatty acid as (S). The involvement in the iron metabolism was not investigated. 

Block (cap) residual lysine residues with a carboxylic acid by acylation using succinic anhydride prepared in l-methyl-2-pyrrolidinone,... 

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