Alkylation of a histidine residue

A different type of reactive bromo compound having a moderate resemblance to hexoses is represented by the bromoconduritols B (40) and F (41), named after the respective parent tetrahydroxycyclohexene. Even thou their hydroxylation pattern resembles that of D-glucose, only a few examples of D-glucosidase inhibition have been reported. The first was a-D-glucosi-dase from yeast, which is inhibited by bromoconduritol B (formerly called bromoconduritol A), having ki(max)/Kj 69,000 M" min", by alkylation of a histidine residue at the active site. 

Irreversible inhibition is probably due to the alkylation of a histidine residue.43 Chymotrypsin is selectively inactivated with no or poor inhibition of human leukocyte elastase (HLE) with a major difference the inactivation of HLE is transient.42,43 The calculated intrinsic reactivity of the coumarin derivatives, using a model of a nucleophilic reaction between the ligand and the methanol-water pair, indicates that the inhibitor potency cannot be explained solely by differences in the reactivity of the lactonic carbonyl group toward the nucleophilic attack 43 Studies on pyridyl esters of 6-(chloromethyl)-2-oxo-2//-1 -benzopyran-3-carboxylic acid (5 and 6, Fig. 11.5) and related structures having various substituents at the 6-position (7, Fig. 11.5) revealed that compounds 5 and 6 are powerful inhibitors of human leukocyte elastase and a-chymotrypsin thrombin is inhibited in some cases whereas trypsin is not inhibited.21... 

P. A. Price, S. Moore, and W- H. Stein. Alkylation of a histidine residue at the active site of bovine pancreatic deoxyribonuclease. J. Biot. Chem. 244 924-928... 

Figure 8. Reactions involved in directing the specificity of bromoacetone to the alkylation of a histidine residue in ficin...

Oxidation of this tryptophan in galactose oxidase also prevents alkylation of the histidine residue. Alkylation of the histidine residue in turn markedly affects the fluorescence quantum yield of this tryptophan (43) and nearly abolishes the absorbance of the copper atom. The copper atom itself is also essential to the reactivity of this histidine. Thus, we appear to have a consistent set of highly interdependent components. Not unexpectedly, the copper site cannot be fully understood without considering its interactions with non-ligand protein groups. 

Crestfield, A.M., Stein, W.H., and Moore, S. (1963) Alkylation and identification of the histidine residues at the active site of ribonuclease. J. Biol. Chem. 238, 2413-2419. 

The facile reaction of CAA and BAA with nucleosides and nucleotides is one example of many of the applications of the bifunctional reactivity of halogenated aldehydes and ketones in modification of biomolecules. In an early example of the extensive use of halogenated ketones as protease substrate analogues, l-V-tosylamido-2-phenylethyl chloro-methyl ketone (TPCK) 30 was synthesized as a chymotrypsin substrate analogue. Stoichiometric inhibition was accompanied by loss of one histidine residue as a result of alkylation by the chloromethyl moiety68. A host of similar analogues were subsequently prepared and used as selective enzyme inhibitors, in particular for the identification of amino acid residues located at enzyme active sites69. 

The above results were confirmed subsequently by Holmes and Stevenson by determining the amino acid sequence and establishing the position of the C-labelled histidine in the peptide B obtained from E3. A reliable amino acid sequence 162 has been obtained for peptide B up to residue 30 which was in agreement with the amino acid sequence determined from the gene sequence of E3. The authors demonstrated that a histidine residue is selectively alkylated within the active site of E. coli E3 when the enzyme is a component of the PD complex. The reagent 161 functions as a unique form of an active-site-directed irreversible inhibitor. 

An illustration of this approach may be seen in the studies on streptococcal proteinase (Liu 1967). The activity of this enzyme is dependent upon the presence of a free sulfhydryl group. The active form of the enzyme was first converted to the inactive S-sulfenyl-sulfonate derivative. Treatment of this derivative with a chemically-reactive substrate "analogue, a-N-bromoacetylarginine methyl ester, resulted in the alkylation of a single histidine residue. The sulfhydryl group of the modified enzyme was regenerated by reduction, however, this did not restore enzymatic activity, thus providing presumptive evidence for the involvement of both a cysteinyl and a histidyl residue in the active site of this enzyme. 

Two other urinary metabolites have been reported in animal studies (Figure 4). N7-(2-Hydroxyethylthioethyl)guanine (6), derived from the breakdown of alkylated DNA, was detected in rats (Fidder et al., 1996). The unusual metabolite (7), assumed to result from reaction with a histidine residue, was identified in the pig following percutaneous administration (Sandelowsky et al., 1992). 

In the past ten years, there has been developed a series of enzyme inhibitors that combine the features of an alkylating agent with specificity for the active site of an enzyme, thus permitting alkylation and identification of a group at or near the active center of an enzyme, or a particular enzyme to be specifically inactivated. Thus a l-chloro-4-phenyl-3-p-toluenesulfonamido-2-butanone ( W-p-tolylsulfonylphenylalanine chloro-methyl ketone ) inactivates chymotrypsin (which cleaves a peptide bond adjacent to an aromatic residue), and 7-amino-l-chloro-3-p-toluene-sulfonamido-2-heptanone ( a-iV-p-tolylsulfonyllysine chloromethyl ketone ) inhibits trypsin (which cleaves a peptide bond adjacent to lysine. In both cases, a histidine residue at the active site is alkylated, and neither inhibitor will inhibit the other enzyme at low concentrations. 

Figure 5. The irreversible inactivation of (3-hydroxydecanoyl thioester dehydrase by A(34) decynoyl N-acetyl cysteamine. The enzyme catalyzes the reversible interconversion of hydroxy-aecanoyl thioesters with their a,(3-trans and B,y-cis counterparts (Scheme 1). The acetylenic analog is converted by the enzyme into the highly reactive conjugated allene, which alkylates a histidine residue in the active center (Scheme 2) (23).

In a paper discussing the alkylation and identification of the histidine residues at the active site of ribonudease, JafTe (1963) has analyzed infrared spectra of the 1-and 3-carboxymethylhistidines and of the 1- and 3-methylhistidines, and has confirmed spectroscopically the positions assigned to the carboxymethyl groups in the... 

Irreversible inhibition can also occur in the presence of halogenated methyl ketones which alkylate the active histidine residue (cf. 2.4.1.1), or as a result of the action of proteinase inhibitors, which are also proteins, by interaction with the enzyme to form inactive complexes. These natural inhibitors are found in the organs of animals and plants (pancreas, colostrum, egg white, potato tuber and seeds of many legumes cf. 16.2.3). The specificity of serine... 

A group of peptide derivatives such as peptide arginals and boronic acid peptide derivatives belong to another class of reversible thrombin inhibitors. One such inhibitor is PPACK (D-Phe-Pro-Arg chloromethyl ketone), which functions as a powerful irreversible thrombin inhibitor by alkylating the histidine residue at the catalytic site of thrombin (58). It, however, is unstable in neutral solution, as it undergoes cyclization and inactivation. However, the D-methyl derivative of D-Phe-Pro-Arg-H (D-Mephe-Pro-Arg-H) called efegatran, with a molecular mass of 515 Da, is a stable selective reversible inhibitor of thrombin with a K. of approximately 100 nM. The basic amino terminus in this compound is responsible for promoting the specificity toward thrombin (63). 

Price et al. (86). The evidence is based on experiments in which DNase I was reacted with iodoacetate at pH 7.2 in the presence of 0.1 M Mn2+. Under these conditions the enzyme is gradually inactivated and the loss of activity parallels the formation of one residue of 3-carboxymethyl histidine per molecule. The rate of the alkylation reaction is dependent on Mn2+ concentration. Substitution of Mn2 by Cu2+ in the presence of tris buffer greatly increases the rate of alkylation. A 29-residue peptide con-... 

The investigations of W. H. Stein and Moore and their colleagues were first reported in 1959 157). The inactivation of RNase by iodo-acetate was studied. A maximum in the rate of activity loss was noted at pH 5.5. Reaction with a methionine residue was found at pH 2.8 at pH 8.5-10 lysine residues were modified, but at pH 5.5-6.0 only histidine appeared to be involved. The specific reaction required the structure of the native enzyme. Reaction with histidine was not observed under a variety of denaturing conditions 158). Iodoacetamide did not cause activity loss, or only very slow loss, or alkylate His 119 in the native enzyme at pH 5.5. The negative charge on the carboxyl group of the iodoacetate ion was apparently essential. 

Alkylation at pH 8.5 shows reduced rates of reaction at the histidine residues but significant substitution at lysine, particularly Lys 41 118). The histidine reactions show the same general stereospecificity as found at pH 5.5. The inactive Lys 41 derivatives (25, 26, and 27 of Table VI) show alkylation patterns of His 12 and 119 at pH 5.5 which are similar to those of RNase-A although with some differences in detail. When Lys 1 and 7 are acetylated in RNase-S the alkylation pattern with iodoacetic acid is not affected. When PIR is used the alkylation of His 119 is nearly abolished but that at His 12 is accelerated 163). The probable interaction of Asp 121 with His 119 may be important in the alkylation reactions observed in the native enzyme and the various lysine derivatives. In PIR this interaction has, of course, been removed. 

Miller et al. have shown that short peptides containing alkylated histidine residues can be used as catalysts for the kinetic resolutions of secondary and some tertiary alcohols. These catalysts are also postulated to effect catalysis by a nucleophilic mechanism. The backbone amides and ancillary functionality are proposed to govern selectivity through catalyst-substrate contacts (e.g., hy-... 

Blood proteins, such as hemoglobin, may be used in tests of human exposure because blood is readily and safely accessible. For example, the exposure of mice to ethylene oxide or dimethylnitrosamine was estimated by measuring alkylated residues in hemoglobin. The method was subsequently extended to people exposed occupationally to ethylene oxide by measuring 7V-3-(2-hydroxycthyl) histidine residues in hemoglobin. Similarly methyl cysteine residues in hemoglobin can be used as a measure of methylation. 

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