Subsite-specific properties

The problem of performing site-specific chemistry at one iron site in a [4Fe-4S] cluster requires interrelating the reactivity of three of the iron atoms such that the fourth site can react independently. This relationship has been achieved by the synthesis of a semirigid tridentate thiolate ligand that readily binds several types of cubane clusters. The details of the synthesis and reactivity of site-differentiated clusters will be provided in Section IV. We next turn to the topic of subsite-specific properties observed in biological systems. 

With subsite-differentiated clusters in hand, we turn to their applications in examining subsite-specific properties of native [4Fe-4S] clusters by the synthetic analogue approach. The potentially most important applications at this stage of development of subsite-specific cluster chemistry are listed below. 

The early substrate series had P,-residues which incorporated a para-nitroanilide in place of the normal scissile amide bond. This feature allowed the kinetics of the hydrolyses to be easily followed spectrophotometrically [95]. As a result, these studies focused on the 8,-85 subsites. 8ome of these substrates, for example (3-4)-(3-6), were based on the sequence of the reactive centre of a,-PI. It was shown that substrate (3-3) which contains a P,-Val is a better substrate than (3-4) which contains the P,-Met found in a,-PI (see Table 2.2). As in a,-PI, oxidation of the P,-Met resulted in a decrease in enzyme recognition, for example, compare (3-4) and (3-5). One of the compounds (3-3) prepared in the course of this work is an excellent, specific substrate for HLE and also has better aqueous solubility than other related substrates. As a result of this combination of properties, compound (3-3) has been used as the standard low-molecular-weight substrate for much of the kinetic analyses of HLE inhibitors, and its peptide backbone has been incorporated into several low-molecular-weight inhibitors. 

To form catalytically productive enzyme/substrate complexes, many peptide bond cis-trans isomerases essentially require the location of the reactive bond of the substrate in the context of secondary binding sites or a specific spatial organization of the polypeptide chain thus creating features of stereo- and regiospecifi-city [19,20]. As in the case of many endoproteases, PPIases can utilize an extended array of catalytic subsites to enhance catalytic efficiency and substrate specificity. These properties precondition peptide bond cis-trans isomerases toward a complex reaction pattern. Consequently, biochemical investigations have led to the elucidation of three distinct molecular mechanisms that might be operative either in isolation or collectively in the cellular action of both prototypical and multidomain peptide bond cis-trans isomerases ... 

A large number of organic compounds reversible or irreversibly inhibits AChE (Long, 1963), which bind either to the esteratic or the anionic subsite of AChE catalytic site or to the peripheral site of the enzymes. Most of them are S5mthetic substances, sometimes with insecticidal properties. Few natural inhibitors of AChE are known and, to date, fasciculins are the only known proteinic AChE inhibitors. They have been shown to display a powerful inhibitory activity toward mammalian AChE. lodination of Fas3 provided a fully active and specific probe of fasciculin-binding sites on rat brain AChE (Marchot et al., 1993). These authors demonstrate that fasciculins bind on a peripheral site of AChE, distinct from the catalytic site and, at least partly, common with the sites on which some cationic inhibitors and the substrate in excess bind since phosphorylation of the catalytic serine (esteratic subsite) by [l,3- H]diisopro-pyl fluorophosphate can still occur on the Fas3. In the... 

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