Protein nucleophile

Cleavage of a peptide bond is an example of a nucleophilic attack. The nucleophile in the reaction is either an activated water molecule or part of the side-chain of an amino acid, and peptidases are described as having either a water nucleophile or a protein nucleophile. Peptidases with a water nucleophile either utilize one or two metal ions as ligands for the water molecule, in which case the peptidase generally acts... 

All peptidases within a family will have a similar tertiary structure, and it is not uncommon for peptidases in one family to have a similar structure to peptidases in another family, even though there is no significant sequence similarity. Families of peptidases with similar structures and the same order of active site residues are included in the same clan. A clan name consists of two letters, the first representing the catalytic type as before, but with the extra letter P , and the second assigned sequentially. Unlike families, a clan may contain peptidases of more than one catalytic type. So far this has only been seen for peptidases with protein nucleophiles, and these clans are named with an initial P . Only three such clans are known. Clan PA includes peptidases with a chymotrypsin-like fold, which besides serine peptidases such as chymotrypsin... 

An affinity label is a molecule that contains a functionality that is chemically reactive and will therefore form a covalent bond with other molecules containing a complementary functionality. Generally, affinity labels contain electrophilic functionalities that form covalent bonds with protein nucleophiles, leading to protein alkylation or protein acylation. In some cases affinity labels interact selectively with specific amino acid side chains, and this feature of the molecule can make them useful reagents for defining the importance of certain amino acid types in enzyme function. For example, iodoacetate and A-ethyl maleimide are two compounds that selectively modify the sulfur atom of cysteine side chains. These compounds can therefore be used to test the functional importance of cysteine residues for an enzyme s activity. This topic is covered in more detail below in Section 8.4. 

The first clue regarding molecules ability to undergo bioactivation, the precursor to MBI, is often determined from its chemical structure. For example, certain substructures are prone to forming reactive intermediates capable of alkylating protein nucleophiles including CYP, as in the case of M BI. A comprehensive look at different chemical structures prone to CYP bioactivation has been reviewed recently [172,173],... 

Chlorotetrolic esters are small, highly functionalized, reactive molecules of particular interest is the possibility of using them as reagents for chemical modification of biological macromolecules. Different protein nucleophiles react under mild conditions with methyl 4-chloro-2-butynoate by addition across the triple bond and/or substitution of chlorine while the triple bond and the ester group are involved in the reaction of chlorotetrolic... 

Primary reaction with sulfhydryl groups and slower reaction with other protein nucleophiles. 

The alkylation of methionine is pH-independent, while that of histidyl, cysteinyl and lysyl residues is pH-dependent, the rate reflecting the concentration of the unprotonated protein nucleophile. 

Valentine et al. (1993) have shown that the initial dithiocarbamate protein adduct decomposes to isothiocyanate derivatives which then react with protein nucleophiles resulting in crosslinking. The crosslinking of protein in the nerve axons to cause their ultimate degeneration is correlated with the crosslinking of spectrin, a blood cell membrane protein. This suggests that the latter can be used as a biomarker of adverse effect of nerve damage. 

Silverman and Zieske have rationalized how a protein nucleophile other than flavin is involved in MAO inactivation reactions, and why different inactivator compounds specifically react with flavin, protein amino acids, or both (100). Hydrogen atom donation from a cysteine residue to the flavin semiquinone radical would produce a thiyl radical, which could then capture the primary or secondary alkyl radical generated on cyclopropyl ring opening from the amine radical cation of the inactivator. The hydrogen atom abstraction reaction between the flavin and active site amino acid may be an equilibrium process such that either species could be present at any turnover. Hence, a combination of steric constraints and proximity to either the flavin semiquinone radical or the thiol radical will determine the site of adduct formation for a particular inactivator structure. A two-dimensional representation is shown in Scheme 23 (compounds 40-42), which illustrates the proposed equilibrium between the flavin semiquinone radical and amino acid as well as the proposed intermediates for the inactivation of MAO by A-(l-methylcyclopropyl)benzylamine 40 (104), rrradical center relative to the particular protein radical is consistent with proposed site of attachment of inactivator to protein 40 is near the flavin radical, such that exclusive flavin attachment occurs, 41 is positioned closer to the amino... 

Figure 7.3. The mechanism proposed for P450 bioactivation of the thiophene in tienilic acid to a reactive species that binds covalently to the protein (the protein nucleophile is denoted by Protein-XH). The structures of two other thiophene-containing drugs, ticlopidine and clopidogrel, that might be similarly activated are also shown.

UGT-catalyzed glucuronidation of the carboxylic acid group in drugs results in the formation of acyl glucuronides, which are intrinsically electrophilic in nature. Protein modification can occur via a simple transacylation reaction with a protein nucleophile(s) or by acyl migration within the f3-0-glucuronide unit to a reactive aldehyde intermediate. Detailed mechanistic discussion on this issue is provided with the NSAIDs ibufenac and ibuprofen in the following section. 

Selective reaction with 4-thio-U resi- 79, 114 due on certain tRNA molecules, and of reactive protein nucleophiles... 

Spectrum of reactivity. All protein nucleophiles can be alkylated by this type of reagent (Table I). Evidence for aflSnity labeling of cysteine, histidine, lysine " and a-amino groups, tyrosine, methionine serine, and glutamic acid has been reported, and it is likely that aspartic acid and threonine can also be labeled. Such a wide spectrum of reactivity is important for successful labeling of residues at the reactive site. 

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