Enzymes peptide sequencing

Crystallographic studies imply that although little sequence homology exists between the different protease cleavage sites, what is conserved is the shape that they adopt within the active site of the enzyme (Prabu-Jeyabalan et al. 2002). This shape has been termed the substrate envelope and represents the consensus volume overlapping the majority of the substrates. Most likely, HIV-1 protease recognizes a particular peptide sequence as being a substrate by a combination of accessibility and the shape the sequence can adopt. 

Proteases are one of the largest families of enzymes and are involved in a multitude of vital processes. Due to their biological relevance and diversity, multiple fluorescent reporters monitoring their activity have been designed and successfully applied in vitro and in vivo [112-114]. Standard small molecule FRET probes for proteases consist of an amino acid sequence flanked by a FRET pair, consisting of two fluorophores or one fluorophore and a quencher molecule. Upon cleavage of the peptide sequence, the emission of the donor fluorophore is dequenched and the intensity increases whereas the emission of the acceptor decreases and vanishes more or less completely in those cases where the acceptor is fluorescent (see Fig. 6.11). 

Protein farnesyl transferase also can be used to add a geranylazide derivative to a synthetic peptide by incorporating the enzyme recognition sequence CAAX at the C-terminal of any... 

An alternative classification scheme was introduced in 2002 and this divides lantibiotics into two subgroups, class I and class II. This scheme primarily classifies lantibiotics according to their biosynthetic enzymes and sequence homology of their leader peptides. Similar to most biosynthetic pathways in bacteria, the genes for lantibiotic biosynthesis are clustered. They have been designated the generic locus symbol Ian, with a more... 

Following the initial isolation of the Hnl from M. esculenta [33] in which the peptide sequence was established, an overexpressed version of this enzyme (in E. coli) was prepared [41]. This system is not limited for enzyme quantity (as outlined in Sect. 2.3), and can accept a wide range of aromatic, heterocyclic and aliphatic aldehydes, as well as ketones, as substrates. In practical terms, a measure of the degree of enzyme inhibition by substrates is of significant value and for this system this has been quantified for a range of aldehydes, ketones and alcohols [70]. It was deduced that ketones and alcohols are competitive inhibitors, whilst aldehydes are noncompetitive inhibitors. 

Various diastereomeric di-, tri-, and tetrapeptides that carry the sterically demanding trifluoromethyl group instead of the natural a-proton at different positions within these short peptide sequences have been designed, and their stability towards enzymatic hydrolysis has been investigated. The structures of the a-trifluoromethyl (aTfm)-substituted amino acids are shown in Scheme 1. From these studies we gained valuable information on how a-trifluoromethyl-substi-tuted peptides may interact with proteins. The aTfm amino acids used in this study combine the conformational restrictions [49-52] of C -dialkylation with the unique stereoelectronic properties of the fluorine atom and have shown interesting effects on peptide-enzyme interactions [53,54]. 

Figure 4.6. Modulation of peptide conformational equilibrium can be achieved through systematic modifications of peptide sequences that direct receptor interactions toward therapeutic response, and away from untoward effects. A systematic peptide modification may lead to reduced concentrations of peptide conformations susceptible to metabolizing enzymes such as peptidases.

Although Ang I contains the peptide sequences necessary for all of the actions of the renin-angiotensin system, it has little or no biologic activity. Instead, it must be converted to Ang II by converting enzyme (Figure 17-1). Ang I may also be acted on by plasma or tissue... 

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