Massively parallel enzyme kinetics reveals the substrate recognition landscape of the metalloprotease ADAMTS13 Journal Articles uri icon

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abstract

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

authors

  • Kretz, Colin
  • Dai, Manhong
  • Soylemez, Onuralp
  • Yee, Andrew
  • Desch, Karl C
  • Siemieniak, David
  • Tomberg, Kärt
  • Kondrashov, Fyodor A
  • Meng, Fan
  • Ginsburg, David

publication date

  • July 28, 2015