Affinity labelling

Treatment of 9-(/ -D-ribofuranosyluronic acid)adenine with diphenylphosphoro-chloridate and orthophosphate or tripolyphosphate yields (62) and (63), which, although unstable, inhibit rabbit AMP aminohydrolase and pyruvate kinase, respectively, with behaviour characteristic of active-site-specific reagents.98 Adenylate kinases from several sources are inactivated by iV6-[2- and 4-fluorobenzoyl]-adenosine-5 -triphosphates, with kinetics characteristic of active-site labelling, although these compounds were without effect on yeast hexokinase and rabbit pyruvate kinase.99 

Two aldehydic nucleotide derivatives have found use as affinity labels. The magnesium salt of (64), formed by oxidation of ATP with periodate, is a competitive inhibitor of pyruvate carboxylase with respect to [Mg. ATP2-],100 and (65), obtained from the / -anomer of 5-formyluridine-5 -triphosphate on treatment with alkali, is a non-competitive and reversible inhibitor of DNA-dependent RNA polymerase from E. coli.101 In each case, addition of borohydride gives stoicheiometric covalent linkage of the nucleotide to the enzyme, with irreversible inactivation. It is thought that condensation with lysine occurs to give a Schiff s base intermediate, which undergoes subsequent reduction. 

6 -Dithio-bis(inosinylimidodiphosphate) (66) forms mixed disulphides between the 6-thiol group of the purine and cysteine residues on myosin on binding four 

A bromoacetylated derivative of the nonsense codon UpGpA has been synthesized and used to label ribosomal proteins.103 

8-azido-ATP with dansyl chloride in bicarbonate buffer at pH 9.8 affords 2 -0-dansyl- 

8-azido-ATP, as indicated by n.m.r. data in comparison with appropriate standards, and this fluorescent photoaffinity agent has been used for the labelling of adenylate kinase from rabbit muscle,336 and of the catalytic subunit of cAMP-dependent protein kinase.337 8-Azido-AMP, -ADP, and -ATP have been treated with 2, 4, 6-trinitrobenzenesulphonic acid in bicarbonate buffer to form their respective 2, 3 -0-(2, 4, 6-trinitrophenyl) derivatives which are superfluorescent (i.e. exhibiting 

Among alkylating affinity labels, 8-[(4-bromo-2, 3-dioxobutyl)thio] ADP and -ATP (199 n = 2 or 3), formed by successive treatments of the corresponding 8-bromoadenosine phosphates with lithium hydrosulphide and 1, 4-dibromo-butanedione, have been used to label pyruvate kinase from rabbit muscle.355 

Inactivation occurred in a biphasic manner, being faster for (199 n = 3) than for (199 n = 2). A binding site distinct from the active site became labelled also. Treatment of 1, N -etheno-ADP with alkali followed by carbon disulphide affords 1, N -etheno- 

2- thio-ADP, which on alkylation with 1, 4-dibromobutanedione gives 2-[(4-bromo-2, 

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Affinity labeling Affinity labels Affinity values Affirm Aflas... 

Herbicidal Inhibition of Enzymes. The Hst of known en2yme inhibitors contains five principal categories group-specific reagents substrate or ground-state analogues, ie, rapidly reversible inhibitors affinity and photo-affinity labels suicide substrate, or inhibitors and transition-state, or reaction-intermediate, analogues, ie, slowly reversible inhibitors (106). 

Affinity Labels. Active site-directed, irreversible inhibitors or affinity labels are usually substrate analogues that contain a reactive electrophilic functional group. In the first step, they bind to the active site of the target enzyme in a reversible fashion. Subsequentiy, an active site nucleophile in close proximity reacts with the electrophilic group on the substrate to form a covalent bond between the enzyme and the inhibitor, typically via S 2 alkylation or acylation. Affinity labels do not require activation by the catalysis of the enzyme, as in the case of a mechanism-based inhibitor. 

The often fast binding step of the inhibitor I to the enzyme E, forming the enzyme inhibitor complex E-I, is followed by a rate-determining inactivation step to form a covalent bond. The evaluation of affinity labels is based on the fulfillment of the following criteria (/) irreversible, active site-directed inactivation of the enzyme upon the formation of a stable covalent linkage with the activated form of the inhibitor, (2) time- and concentration-dependent inactivation showing saturation kinetics, and (3) a binding stoichiometry of 1 1 of inhibitor to the enzyme s active site (34). 

A good example of an affinity label for creatine kinase has been presented (35). This enzyme catalyzes the reversible transfer of a phosphoryl group from adenosine triphosphate [56-65-5] (17) to creatine [57-00-1] (18), leading to adenosine diphosphate [7584-99-8] (19) and phosphocreatine [67-07-2]... 

A/-(2,3-Epoxypropyl)-A/-amidinoglycine [70363-44-9] (21) was shown to be an affinity label of creatine kinase. Its mechanism of covalent bond formation is outlined as follows ... 

Berg R. H., G. W. Erdos, M. Gritzali and R. D. Brown. (1988). Enzyme-gold affinity labeling of cellulose. Journal of Electron Microscopy Techniques 8 371-379. 

The third mechanism that results in slow binding behavior is covalent inactivation of the enzyme by affinity labeling or mechanism-based inhibition (Scheme... 

Figure 8.2 Mechanisms of irreversible enzyme inactivation. (A) Nonspecific affinity labeling, (B) quiescent affinity labeling, and (C) mechanism-based inactivation.

Depending on the mechanism of irreversible reaction, inactivation can appear to proceed through either a single-step or a two-step mechanism (Figure 8.2). In the case of nonspecific affinity labels (see Section 8.2) many amino acid residues on the enzyme molecule, and on other protein molecules in the sample, can be covalently modified by the affinity label. Not every modification event will lead to inactiva-... 

Thus, at concentrations of inactivator well below Kh a plot of kohs as a function of [/] will be linear, and the slope of the line will be equal to k JK This is exactly the case that we encountered above for nonspecific affinity labeling. 

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. 

However, the conversion of omeprazole to the active sulphenamide does not result in formation of a reversible enzyme inhibitor, but rather results in in situ formation of a powerful affinity label. Hence we can consider omeprazole to be a unique example of quiescent affinity labeling in which selectivity results from the unique environment of the target enzyme. 

More recently attempts to generate highly selective quiescent affinity labels have been made for a number of protease and kinase targets. As examples, inhibitors of the Rhinovirus 3C protease (Mathews et al 1999) and of the epidermal growth factor receptors (Boschelli, 2002), both incorporating Michael acceptors to covalently inactivate cysteine residues in their target enzymes (Lowry and Richardson, 1981 Figure 8.6), have entered human clinical trials for the treatment of rhinovirus infection and cancer, respectively. 

The lack of target enzyme specificity is a critical liability for the use of affinity labels as drugs. The inherent chemical reactivity of these compounds almost ensures that... 

Table 8.1 Some examples of quiescent affinity labels of clinical interest...

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