Massively parallel, computationally guided design of a proenzyme

Brahm J. Yachnin, Laura R. Azouz, Ralph E. White, Conceição A.S.A. Minetti, David P. Remeta, Victor M. Tan, Justin M. Drake, Sagar D. Khare

Research output: Contribution to journalArticlepeer-review

6 Scopus citations


Confining the activity of a designed protein to a specific microenvironment would have broad-ranging applications, such as enabling cell type-specific therapeutic action by enzymes while avoiding off-target effects.While many natural enzymes are synthesized as inactive zymogens that can be activated by proteolysis, it has been challenging to redesign any chosen enzyme to be similarly stimulus responsive. Here, we develop a massively parallel computational design, screening, and next-generation sequencing-based approach for proenzyme design. For a model system, we employ carboxypeptidase G2 (CPG2), a clinically approved enzyme that has applications in both the treatment of cancer and controlling drug toxicity. Detailed kinetic characterization of the most effectively designed variants shows that they are inhibited by ∼80% compared to the unmodified protein, and their activity is fully restored following incubation with site-specific proteases. Introducing disulfide bonds between the pro- and catalytic domains based on the design models increases the degree of inhibition to 98% but decreases the degree of restoration of activity by proteolysis. A selected disulfide-containing proenzyme exhibits significantly lower activity relative to the fully activated enzyme when evaluated in cell culture. Structural and thermodynamic characterization provides detailed insights into the prodomain binding and inhibition mechanisms.

Original languageEnglish (US)
Article numbere2116097119
JournalProceedings of the National Academy of Sciences of the United States of America
Issue number15
StatePublished - Apr 12 2022

Bibliographical note

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© 2022 National Academy of Sciences. All rights reserved.


  • Rosetta
  • carboxypeptidase G2
  • proenzyme design
  • protein design
  • zymogens


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