Affinity labels, nonspecific

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. 

Since the discovery that glycolate was an alternate substrate for pyruvate kinase ( ), several other o-hydroxy acids have also been found to be substrates for this enzyme ( ). This class of alternate substrates provides a new approach the problem of substrate specificity for pyruvate kinase. 3-Nitrolactate is one such alternate substrate. Interestingly, the phosphorylated product of this reaction inactivates the enzyme (86). However, 3-nitrolac-tate does not behave as a straightforward affinity label since covalent modification occurs nonspecifically. It is hoped that this new Information may lead to the design of an affinity label of this enzyme, further serving to pinpoint amino acid groups at the active site. 

Second, the enzyme is covalently bound to the affinity label in conditions chosen according to the enzyme in question and the chemical nature of the analogue, so as to decrease nonspecific labeling. Third, the labeled enzyme is subjected to proteolysis and the radioactive peptide(s) are isolated by HPLC. The labeled peptide(s) are then sequenced, providing information about the domains of the enzyme involved in the interaction with the substrate (or with the modulators). 

In these cases, as with affinity labels, nonspecific covalent modification of residues other than those located in the active site cannot be excluded. A second test for a metabolically activated affinity label is to add an additional aliquot of fresh enzyme to the incubation buffer. The fresh enzyme should be inactivated at a higher rate than that of the first equivalent of enzyme because there is more reactive species present in solution. By contrast, the mechanism-based inhibitor should show no difference in rate until the concentration of inhibitor is depleted. It should also be noted that the observation of such rate increases necessitates that the reactive species is relatively stable and is not immediately quenched by the incubation buffer. 

Alkyl halides are even less reactive than acyl halides, as indicated by the compilation of reaction rates of thiolate anions with various types of alkyl halides (282). Nevertheless, potentially useful affinity labels have been synthesized with alkyl halide substituents and have been shown to specifically inactivate several enzymes, albeit slowly the low reactivity of the alkyl halides may minimize nonspecific reaction. Adenosine 5 -(2-bromoethyl)phosphate has been characterized and reported to inactivate NAD -dependent isocitrate dehydrogenase (283). The 2 - and 3 -(2-bromoethyl)-AMP labels have also been synthesized, and model reactions of the bromoethyl-AMPs with cysteine, lysine, histidine, and tyrosine have been studied (284). More recently, esters of adenosine 5 -monophosphate have been prepared with ethyl, propyl, or hexyl moieties and bromo or chloro substituents at the w position (285). Yeast alcohol dehydrogenase exhibited enhanced inactivation by the hexyl derivative, but inactivation rates of other dehydrogenases were unremarkable. Two iodopropyl derivatives of cAMP have been described, namely, 1, A -(3-iodopropyleno)adenosine 3, 5 -cyclic monophosphate and 3 -0-(2-iodo-3-hydroxypropyl)adenosine 3, 5 -cyclic monophosphate the latter inactivates cAMP phosphodiesterase from human platelets, with a pseudo-first-order rate constant of 0.147 hr" (286). 

It is important to have experimental criteria by which to judge whether an inhibitor requires catalytic conversion and whether direct inactivation of the enzyme occurs. The following criteria may be used to decide these points (1) The kinetics of inactivation should be first order in inhibitor concentration. (2) The pH vs. rate profile for inhibition may be similar to that for substrate turnover. (3) If C—bonds are cleared, a deuterium isotope effect should be manifest during the inhibition. (4) Trapping agents, such as mercaptans, should not decrease the rate of inactivation by inhibitor. The rate would decrease if the reactive molecule first diffused into solution and only later inactivated the enzyme by an affinity labeling or nonspecific mode of inactivation. (5) If a second aliquot of fresh enzyme is added to a solution containing inactivated enzyme and excess inhibitor, the rate of inactivation of the second... 

Limitations. Isothiocyanates react rather rapidly with exposed amino groups, whether or not they are attached to aflSnity groups. Therefore, nonspecific reaction can be expected to occur, and the investigator must demonstrate that the effects he observes with isothiocyanate affinity labels are indeed specific effects rather than the result of nonspecific interaction. 

Complex receptors typically have a multiplicity of reactive residues outside the ligand binding site. Nonspecific reaction of affinity labels with these groups can be a serious problem. Some of these residues are likely to be accessible only to small molecules. Hence, the amount of nonspecific labeling will be less when a macromolecular probe is used. 

To minimize nonspecific covalent reaction during the product analysis procedures, we routinely terminate <-BrAcLys-tRNA affinity labeling incubations by adding 2-mercaptoethanol to 0.1 M and incubating for 15 min at 37°. Nonspecific reactions can be minimized in photoaflfinity labeling experiments by the use of scavenger molecules. ... 

In contrast to this sensitive and highly selective response of pyruvate kinase to affinity labeling of a small number of lysine residues, lactate dehydrogenase loses activity exactly in proportion to the fraction of total lysines in the molecule converted to homoarginine with the nonspecific reagent 0-methylisourea. As would also be expected of an affinity labeling process, the rate of reaction of DMPA with the specific lysines in the catalytic sites of pyruvate kinase is very fast compared with the rate of reaction for a nonaffinity process, the former being of the order of 2500 times faster than the latter. ... 

Becker et al. specifically inactivated the biotin transport system of E. coli using biotin p-nitrophenyl ester. The lactose transport protein of E. coli was labeled by AT-bromoacetyl-/8-D-galactopyranosylamine. In the only reported attempt to isolate an affinity labeled protein, glucose 6-isothiocyanate, which is an affinity label for the glucose transport system in human erythrocytes, gave enough nonspecific labeling of other membrane proteins to render identification of the transport protein difficult. 

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