What question did this study set out to answer?

This research aims to synthesize active ribonuclease A using a designed peptide ligase with unnatural catalytic residues.

October 14, 1994Science

A Designed Peptide Ligase for Total Synthesis of Ribonuclease A with Unnatural Catalytic Residues

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Randomized trial demonstrates peptide ligase efficacy for ribonuclease A synthesis, highlighting novel catalytic mutations.

This research aims to synthesize active ribonuclease A using a designed peptide ligase with unnatural catalytic residues.
Used engineered subtilisin BPN' (subtiligase) for peptide ligation in aqueous solution.
Synthesis involved stepwise ligation of six esterified peptide fragments.
Created variants of RNase A with substituted catalytic histidines using 4-fluorohistidine.
Fully active RNase A (124 residues) produced in milligram quantities with 70% average yield per ligation.
Observed large changes in pH profile affecting reaction rates with minimal change in kcat for RNA cleavage.
Indicated that imidazoles function as general acids and bases with correct protonation states.

Jackson et al. (1994) studied this question.

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