Massively parallel enzyme kinetics reveals the substrate recognition landscape of the metalloprotease ADAMTS13

• DOI: 10.1073/pnas.1511328112
• Publication Date: 2015-07-14T02:45:12Z
• Authors: Colin A. Kretz; Manhong Dai; Onuralp Soylemez; Andrew Yee; Karl C. Desch; David Siemieniak; Kärt Tomberg; Fyodor A. Kondrashov; Fan Meng; David Ginsburg
• Keywords: ADAMTS13; Cleavage (geology); Cleave

Significance Here we report a method to rapidly examine the effect of nearly all possible single amino acid substitutions within substrate fragment coagulation protein von Willebrand factor (VWF) on efficiency cleavage by its cognate protease, ADAMTS13. A phage display library was generated containing ∼3.5 × 10 7 independent clones and uncleaved phages collected at multiple reaction time points after with Analysis these high-throughput sequencing facilitated simultaneous calculations k cat / K M values for each position this fragment, providing comprehensive picture recognition landscape interaction between ADAMTS13 VWF. This approach should be broadly applicable many other protease/substrate pairs.