Active site peptide profiling

Okerberg ES, Wu J, Zhang B et al (2005) High-resolution functional proteomics by active-site peptide profiling. Proc Natl Acad Sci USA 102 4996-5001... 

J Grandchamps, M Nguyen-Disteche, C Damblon, JM Frere. Streptomyces K15 active-site serine DD-transpeptidase specificity profile for peptide, thiol ester carbonyl donors and pathways of the transfer reactions. Biochem J 307 335-339, 1995. 

Figure 14 Profiling of aspartyl protease activity, (a) Catalytic mechanism of aspartyl proteases that cleave peptide bonds via activation of a water molecule, (b) A hydroxyethylene dipeptide isostere-based probe binds tightly to the active site and gets covalently attached via UV irradiation.

The ready availability of amino acids and their different functionalizations in the side chains allowed for a number of applications in the field of supported catalysis. While the relatively low cost of many amino acids apparently does not seem to justify the preparation of supported catalysts derived from amino acids, other reasons (as mentioned above) may drive towards the immobilization of chiral catalysts, for example to experiment with different solubilities, the easy separation of the product from the catalyst, and the catalyst s recyclability. The immobilization of these compounds on a support can also be seen as an attempt to develop a minimalist version of an enzyme, with the amino acid playing the role of the enzyme s active site and the polymer that of an oversimplified peptide backbone not directly involved in the catalytic activity [34]. It should be mentioned at this point that, in principle, amine-based catalysts offer also the possibility to be recovered by exploiting their solubility profiles in acids. 

Hirudin is produced by the medicinal leech and may be extracted from its saliva. It is a 65 amino acid peptide. It is now expressed recombinantly in yeast cells and the recombinant product is almost identical to the naturally occurring protein. It acts directly on thrombin, binding to the active site of thrombin, and preventing the enzymatic cleavage of fibrinogen. It will bind both to free thrombin and to thrombin involved in complexes with fibrinogen. It has the advantage that it does not have the same side effect profile as heparin and thus can be used in heparin-intolerant patients. Bivalimdin is low MW (2180) synthetic peptide which mimics hirudin and binds to thrombin at the same site. 

Figure 3 shows the final chromatogram and activity profile of purified alpha-endopsychosin. The first peak contained most of the PCP displacing activity as measured by its ability to inhibit 3H-PCP. An aliquot of the most active material was hydrolyzed in acid and the amino acid composition was determined using OPA detection. It was determined that the peptide contained approximately 26 amino acids, in close agreement with the molecular weight predicted by Sephadex gel filtration studies. N-terminal analysis revealed that the peptide was blocked at this site. The nature of this blockade is yet to be determined. Studies are under way to determine the amino acid sequence of the peptide. 

Lue, R. Y, Zhu, Q., Li, D. (2004). Site-specific peptide immobilization strategies for the rapid detection of kinase activity on microarrays. Site-specific immobilization of biotinylated proteins for protein microarray analysis. Enzymatic profiling system in a small-molecule microarray. Intein-mediated biotinylation of proteins and its application in a protein microarray. Developing site-specific immobilization strategies of peptides in a microarray. Methods Mol. Biol. 278, 191-204. 

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