Enzymes, simple under appropriate enzyme

Pyruvate formate-lyase activating enzyme is the member of the radical-SAM family whose cluster properties are most similar to those of aconitase. The cluster in pyruvate formate-lyase activating enzyme is quite labile, and in fact until 1997 it was not known that the enzyme contained an iron-sulfur cluster, as all preparations to that time had been done aerobically, under which conditions the cluster falls apart. It was initially reported that PFL-AE contained a mixture of [2Fe-2S] and [4Fe-4S] clusters, and subsequent reconstitution studies of the apo enzyme provided evidence for a [4Fe-4S] cluster. Further studies showed that anaerobic isolation resulted in purification of a form of PFL-AE that contained primarily [3Fe-4S] clusters, which upon reduction converted to [4Fe-4S] clusters.This reductive cluster conversion from [3Fe S] to [4Fe-4S] clusters even in the absence of added iron was remarkably reminiscent of aconitase (see Section 8.27.2.2), and suggested a labile cluster site. Adding to the similarity to aconitase, Mossbauer spectroscopy provided evidence for a linear [3Fe-4S] cluster in PFL-AE isolated under appropriate conditions.Therefore all of the cluster forms previously identified in aconitase were also found in PFL-AE, and like aconitase it appeared to be relatively simple to interconvert between these cluster forms. 

Determination of Extent of Reaction of 3 - and 5 -FSBA with Proteins. There are three relatively simple methods to detect the extent of reaction of these adenosine analogs with proteins. First, the release of fluoride ion from the compound can be followed with a specific fluoride electrode during incubation with a known amount of protein. Such data can be used to estimate the number of sites on the protein that have been modified, providing appropriate corrections are made for any reaction between the compounds and the buffer under the same conditions. Second, the extent of incorporation of sulfonylbenzoyladenosine groups can be approximated from the difference in absorbance at 259 nm between the isolated modified enzyme and native enzyme, using the extinction coefficient of free 3 - or 5 -FSBA at that wavelength. Third, and most directly, the number of moles of reagent incorporated per mole of protein can be determined with radioactive 3 - or 5 -FSBA. 

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