Substrates, specificity modification

Applications of peroxide formation are underrepresented in chiral synthetic chemistry, most likely owing to the limited stability of such intermediates. Lipoxygenases, as prototype biocatalysts for such reactions, display rather limited substrate specificity. However, interesting functionalizations at allylic positions of unsaturated fatty acids can be realized in high regio- and stereoselectivity, when the enzymatic oxidation is coupled to a chemical or enzymatic reduction process. While early work focused on derivatives of arachidonic acid chemical modifications to the carboxylate moiety are possible, provided that a sufficiently hydrophilic functionality remained. By means of this strategy, chiral diendiols are accessible after hydroperoxide reduction (Scheme 9.12) [103,104]. 

PK Banerjee, GL Amidon. Physicochemical property modification strategies based on enzyme substrate specificities I Rationale, synthesis, and pharmaceutical properties of aspirin derivatives. J Pharm Sci 70 1299, 1981. 

The substrate specificity of these enzymes is not stringent for example, CCD1 from tomato was also shown to cleave at the 9,10- and 9, 10 -positions of (1-carotene, zeaxanthin, lutein, violaxan-thin and neoxanthin all of which have different ionone ring modifications. Unlike NCEDs, CCD1 enzymes have no plastid-targeting sequences and are localized in the cytosol. It is postulated that they access the carotenoids in the plastids through a monotopic membrane association (Kloer et al. 

Hemoglobin is another heme-containing protein, which has been shown to be active towards PAH, oxidation in presence of peroxide [420], This protein was also modified via PEG and methyl esterification to obtain a more hydrophobic protein with altered activity and substrate specificity. The modified protein had four times the catalytic efficiency than that of the unmodified protein for pyrene oxidation. Several PAHs were also oxidized including acenaphthene, anthracene, azulene, benzo(a)pyrene, fluoranthene, fluorene, and phenanthrene however, no reaction was observed with chrysene and biphenyl. Modification of hemoglobin with p-nitrophenol and p-aminophenol has also been reported [425], The modification was reported to enhance the substrate affinity up to 30 times. Additionally, the solvent concentration at which the enzyme showed maximum activity was also higher. Both the effects were attributed to the increase in hydrophobicity of the active site. 

In addition to containing protein-protein interaction motifs, E3-substrate specificity may be affected by post-translational modifications. In particular, phosphorylation can alter E3-substrate interactions. One example is p53 where certain phosphorylations inhibit its direct binding to Mdm2, while others indirectly enhance their association by promoting nuclear localization of p53 [104-106]. Phosphorylation also directly enhances substrate interactions, as exemplified by the Cbls, which include phospho-tyrosine binding domains (see below) [107]. 

Ubiquitin ligases largely control the substrate specificity of ubiquitin-conjugation reaction. The temporal specificity of ubiquitin conjugation to substrates by these enzymes is provided by regulation of the ligase activity. Activity of ubiquitin ligases can be modulated by posttranslational modification such as phosphorylation and by allosteric modification of the enzyme, or by attachment to UbL proteins. 

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. 

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