Histidine acylation

Ynenol lactones are also proposed to inactivate serine proteases irreversibly by alkylation of the active site histidine. Acylation of elastase by ynenol lactones produces an electrophilic allenone intermediate (Fig. 51) which covalently modifies and inactivates the enzyme with a partition ratio of 1.7 (Copp et al., 1987). Direct addition of the allenone carboxylic acid is without effect, demonstrating that the inactivator must be tethered in the active site to allow reaction with the enzyme. Substitution a to the lactone carbonyl is required for loss of activity, whereas the rate of inactivation is decreased by substitution at the acetylene terminus, suggesting that allene formation is slowed or that nucleophilic attack on the allene is hindered. 

The metabolic breakdown of triacylglycerols begins with their hydrolysis to yield glycerol plus fatty acids. The reaction is catalyzed by a lipase, whose mechanism of action is shown in Figure 29.2. The active site of the enzyme contains a catalytic triad of aspartic acid, histidine, and serine residues, which act cooperatively to provide the necessary acid and base catalysis for the individual steps. Hydrolysis is accomplished by two sequential nucleophilic acyl substitution reactions, one that covalently binds an acyl group to the side chain -OH of a serine residue on the enzyme and a second that frees the fatty acid from the enzyme. 

The tetrahedral intermediate expels a diacylglycerol as the leaving group and produces an acyl enzyme. The step is catalyzed by a proton transfer from histidine to make the leaving group a neutral alcohol. 

Steps 3-4 of Figure 29.2 Hydrolysis The second nucleophilic acyl substitution step hydrolyzes the acyl enzyme and gives the free fatty acid by a mechanism analogous to that of the first two steps. Water is deprotonated by histidine to give hydroxide ion, which adds to the enzyme-bound acyl group. The tetrahedral... 

This intermediate expels a dtacylglycerol as leaving group in a nucleophilic acyl substitution reaction, giving an acyl enzyme. The dtacylglycerol is protonated by the histidine cation. 

The tetrahedral intermediate expels the serine as leaving group in a second nucleophilic acyl substitution reaction, yielding a free fatty acid. The serine accepts a proton from histidine, and the enzyme has now returned to its starting structure. 

Figure 29.2 MECHANISM Mechanism of action of lipase. The active site of the enzyme contains a catalytic triad of aspartic acid, histidine, and serine, which react cooperatively to carry out two nucleophilic acyl substitution reactions. Individual steps are explained in the text.

Step 3 of Figure 29.3 Alcohol Oxidation The /3-hydroxyacyl CoA from step 2 is oxidized to a /3-ketoacyl CoA in a reaction catalyzed by one of a family of L-3-hydroxyacyl-CoA dehydrogenases, which differ in substrate specificity according to the chain length of the acyl group. As in the oxidation of sn-glycerol 3-phosphate to dihydroxyacetone phosphate mentioned at the end of Section 29.2, this alcohol oxidation requires NAD+ as a coenzyme and yields reduced NADH/H+ as by-product. Deprotonation of the hydroxyl group is carried out by a histidine residue at the active site. 

The mechanism for the lipase-catalyzed reaction of an acid derivative with a nucleophile (alcohol, amine, or thiol) is known as a serine hydrolase mechanism (Scheme 7.2). The active site of the enzyme is constituted by a catalytic triad (serine, aspartic, and histidine residues). The serine residue accepts the acyl group of the ester, leading to an acyl-enzyme activated intermediate. This acyl-enzyme intermediate reacts with the nucleophile, an amine or ammonia in this case, to yield the final amide product and leading to the free biocatalyst, which can enter again into the catalytic cycle. A histidine residue, activated by an aspartate side chain, is responsible for the proton transference necessary for the catalysis. Another important factor is that the oxyanion hole, formed by different residues, is able to stabilize the negatively charged oxygen present in both the transition state and the tetrahedral intermediate. 

The P-alanyl dipeptides carnosine and anserine (A -methylcarnosine) (Figure 31-2) activate myosin ATPase, chelate copper, and enhance copper uptake. P-Alanyl-imidazole buffers the pH of anaerobically contracting skeletal muscle. Biosynthesis of carnosine is catalyzed by carnosine synthetase in a two-stage reaction that involves initial formation of an enzyme-bound acyl-adenylate of P-alanine and subsequent transfer of the P-alanyl moiety to L-histidine. 

After the nucleophilic attack by the hydroxyl function of the active serine on the carbonyl group of the lactone, the formation of the acyl-enzyme unmasks a reactive hydroxybenzyl derivative and then the corresponding QM. The cyclic structure of the inhibitor prevents the QM from rapidly diffusing out of the active center. Substitution of a second nucleophile leads to an irreversible inhibition. The second nucleophile was shown to be a histidine residue in a-chymotrypsin28 and in urokinase.39 Thus, the action of a functionalized dihydrocoumarin results in the cross-linking of two of the most important residues of the protease catalytic triad. 

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). 

In related work a library of 1,458 peptide ligands and various metal salts was tested in hydrolysis reactions of (p-nitrophenyl)phosphates.35 An active substructure composed of polymer-bound histidine in combination with Eu3+ was identified by further dissecting the original hit structure. It needs to be pointed out that catalytically active polymer beads can also be tested for catalytic activity using IR-thermography. In a seminal paper this was demonstrated using 7,000 encoded polymer beads prepared by split-and-pool methods, specifically in the metal-free acylation of alcohols.36... 

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). 

Analysis of deacylation by histidinyl-functionalized micelles suggests that the histidinyl group can act both nucleophilically, generating an acylated histidine intermediate, and as a general base. These conclusions are consistent with the kinetic solvent hydrogen isotope effect (Murakami et al., 1981). 

FIGURE 6.10 The side chain of histidine is readily acylated (A) by activated residues. The imidazolide produced is an activated species similar to the intermediate generated by reaction (B) of a carboxylic acid with coupling reagent carbonyldiimidazole. (Staab, 1956). Imida-zolides acylate amino and hydroxyl groups. Isomerization of histidyl during activation results from abstraction (C) of the a-proton by the 7t-nitrogen. 

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. 

Other serine hydrolases such as cholinesterases, carboxylesterases, lipases, and fl-lactamases of classes A, C, and D have a hydrolytic mechanism similar to that of serine peptidases [25-27], The catalytic mechanism also involves an acylation and a deacylation step at a serine residue in the active center (see Fig. 3.3). All serine hydrolases have in common that they are inhibited by covalent attachment of diisopropyl phosphorofluoridate (3.2) to the catalytic serine residue. The catalytic site of esterases and lipases has been less extensively investigated than that of serine peptidases, but much evidence has accumulated that they also contain a catalytic triad composed of serine, histidine, and aspartate or glutamate (Table 3.1). 

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