Affinity labeling, analogs

Affinity Labeling with UDP-pyridoxal. Read and Delmer (21) utilized the substrate analog UDP-pyridoxal to inhibit mung bean glucan synthase. This affinity label inhibited glucan synthase at micromolar levels and inhibition was protected against with UDP-glucose. A 42 kD polypeptide could be labelled with [3H]UDP-pyridoxal. 

Halomethyl ketones and acids are known to react with thiols and imidazoles. TPCK reacts far more rapidly with chymotrypsin than it does with normal histidine-containing peptides because of its high reactivity as an affinity label. This can be seen in Table 9.2 for an analogous chloromethyl ketone. In addition to this important diagnostic feature, the irreversible inhibition of chymotrypsin by TPCK has four other characteristic features 1,4... 

This effect can be reduced if affinity-labeling kinetic data are analyzed by other types of linear plots used by enzyme kineticists. Two which appear to be useful improvements over the Kitz-Wilson plot are analogs of the Eadie-Hofstee and the Eisenthal-Comish-Bowden plots (20,21). 

This expression for kohB is of the same mathematical form as the analogous expression for the effects of [R] and [L] in true affinity labeling (see Equation 19). In other words, protection by [L] is quantitatively the same for both mechanisms and thus cannot differentiate between them. 

Derivatives of fentanyl and sufentanil with chemo- and photoaffinity functionalities attached to the anilido nitrogen (e.g., NCOCH2Br, NCOCH=N2) have been reported, but affinity labeling experiments performed with theses compounds on brain tissue met with little success. Some of the compounds, notably the diazoacetyl analog of cis 3-methylfentanyl, displayed high binding affinities.(60)... 

Photoaffinity labels have also been investigated. These are enkephalin analogs containing a photosensitive group [e.g., 4-azidophenyl or 2-nitro-4-azidophenyl (NAP)] that form a covalent bond upon irradiation with UV light. Thus, both the peptide 11<37) and the Leu-enkephalins 12(38) bind strongly to brain membranes on photolysis, and the latter pair have been shown to cause a 20-30% inactivation of opioid receptors. Further work on affinity labels and related topics is described in the 1982 and 1983 reports of the International Narcotics Research Conference/39 ... 

Although the organic modifiers are usually not specific for a given enzyme, the second group, the affinity labels, have a degree of specificity built in. Sometimes described as active-site directed, irreversible inhibitors, affinity labels are usually substrate or product analogs that contain an additional chemically reactive moiety. They first bind to the en-... 

Affinity labels are molecules that are structurally similar to the substrate for the enzyme that covalently modify active site residues. They are thus more specific for the enzyme active site than are group-specific reagents. Tosyl-l-phenylalanine chloromethyl ketone (TPCK) is a substrate analog for chymotrypsin (Figure 8.21). TPCK binds at the active site and then reacts irreversibly with a histidine residue at that site, inhibiting the enzyme. The compound 3-bromoacetol is an affinity label for the enzyme triose phosphate isomerase (TIM). It mimics the normal substrate, dihydroxyacetone phosphate, by binding at the active site then it covalently modifies the enzyme such that the enzyme is irreversibly inhibited (Figure 8.22). 

---