1. The goal: analyze complex enzymatic pathways How do you increase the specificity of enzyme detection?

2. The specificity problem In order to be useful for global enzymatic analysis, each microarray spot should be as specific as possible to just one unique enzyme Short peptides, commonly used for enzymatic analysis, have a lot of cross-talk between different enzymes Need general methods to improve the specificity of enzymatic targets

3. How to increase the specificity of synthetic enzyme targets? Problem is that short peptide substrates don’t have enough binding specificity Binding specificity determined by more than just enzyme’s active site Need to make substrates that are sensitive to these other “steric” factors

4. Thrombin substrate insights Previous workers studied a number of different thrombin protease substrates, coupled to different fluorescent groups The peptide “Tos-Gly-Pro-Arg” has a different enzymatic specificity depending upon which fluorescent group was used Fluorescent group is acting as a “steric restrictor” helping to confer additional specificity onto the substrate peptide. Generalize this finding to “steric restrictor” concept

5. Conventional enzyme substrates Enzyme 1 Substrate 1 Enzyme 2 Substrate 2 Cross-reaction Conventional substrates are too small to fully distinguish between enzyme types

6. Steric restrictor concept: Start with a substrate peptide, and modify it to increase specificity Specificity is increased by the addition of two or more carefully optimized “steric restrictor groups”. D-amino acids are good for this: good variation & protease resistant Enzyme Substrate

7. MP’s high specificity substrates Additional “steric restrictor” groups minimize cross-reactions

8. Selecting for high specificity Candidate substrates are bound to a solid phase support, and screened for (-) reaction for non-target enzymes, and (+) reaction with target enzymes.

9. High specificity substrate application 20030113768 A1 Use of “steric restrictor groups” to enhance enzymatic specificity for all peptide enzymatic substrates Specific claims on types of steric restrictor groups Specific claims on methods to generate steric restrictor groups