Artificial Zymogen Based on Protein-Polymer Hybrids

Abstract

This study explores the synthesis and application of artificial zymogens using protein-polymer hybrids to mimic the controlled enzyme activation observed in natural zymogens. Pro-trypsin (pro-TR) and pro-chymotrypsin (pro-CT) hybrids were engineered by modifying the surfaces of trypsin (TR) and chymotrypsin (CT) with cleavable peptide inhibitors utilizing surface-initiated atom transfer radical polymerization. These hybrids exhibited 70 and 90% reductions in catalytic efficiency for pro-TR and pro-CT, respectively, due to the inhibitory effect of the grafted peptide inhibitors. The activation of pro-TR by CT and pro-CT by TR resulted in 1.5- and 2.5-fold increases in enzymatic activity, respectively. Furthermore, the activated hybrids triggered an enzyme activation cascade, enabling amplification of activity through a dual pro-protease hybrid system. This study highlights the potential of artificial zymogens for therapeutic interventions and biodetection platforms by harnessing enzyme activation cascades for precise control of catalytic activity.

Figure 1

Concept of pro-protease hybrids and details of macromonomers. (A) Pro-TR hybrid synthesized using a macromonomer containing TR inhibitor and CT cleavable sites. (B) Pro-CT hybrid synthesized using a macromonomer containing CT inhibitor and TR cleavable sites.

Scheme 1. Preparation of Pro-Protease Hybrids by “Grafting-from” ATRP Approach

An ATRP initiator was first reacted with amino groups on the CT and TR surfaces. Next, ATRP was used to graft copolymers of CBMA and macromonomers from the enzyme surface. (A) pro-TR hybrid with CT cleavable TR inhibitor, (B) pro-CT hybrid with TR cleavable CT inhibitor.

Figure 2

¹H NMR spectra of pro-protease polymer hybrids. (A) ¹H NMR spectrum of the pro-TR hybrid in D₂O. (B) ¹H NMR spectrum of pro-CT hybrid in D₂O.

Scheme 2. Activation (Cleavage of Inhibitor) of Pro-Protease Hybrids by Trigger Protease

(A) Activation of pro-TR hybrid by native CT. (B) Activation of pro-CT hybrid by native TR.

Figure 3

¹H NMR and SEC traces of pro-protease biohybrids before and after activation. (A-1) ¹H NMR spectrum of the activated pro-TR polymer hybrid by native CT. The red boxes show the proton signals of the hybrid before activation. (A-2) SEC trace of pro-TR hybrid before and after incubation with native CT. (B-1) ¹H NMR analysis of the activated pro-CT polymer hybrid by native TR. The green boxes show the proton signals of the hybrid before activation. (B-2) SEC trace of the pro-CT hybrid before and after incubating with native TR.

Figure 4

Relative enzymatic activity of pro-protease polymer hybrids before and after adding trigger protease. (A-1) Michaelis–Menten parameters toward Tos-Gly-Pro-Arg-pNA for pro-TR hybrid activated by native CT incubation. (A-2) Relative enzymatic efficiency of pro-TR hybrid before and after incubating with native CT. (B-1) Michaelis–Menten parameters toward Suc-Ala-Ala-Pro-Phe-pNA for pro-CT hybrid activated by native TR incubation. (B-2) Relative enzymatic efficiency of pro-CT hybrid before and after incubating with native TR. Data are presented as the mean ± standard deviation (SD) of three experiments. Mean differences between experimental groups were tested with an unpaired t test. Values were significantly different at the *p < 0.05 or **p < 0.01.

Scheme 3. Activation Amplification through Cascade Activity of Artificial Pro-CT and Pro-TR Hybrids by Trypsin Incubation

The red scissors indicate the site of cleavage by activated TR, and the green scissors indicate the site of cleavage by activated CT, respectively.

Figure 5

Increment of relative enzymatic activity of the dual pro-CT and pro-TR hybrid cascade system and single pro-CT hybrid using BSA-macrosubstrate. Dual pro-TR and pro-CT hybrids without native TR trigger treatment (open black circle), single pro-CT hybrid with native TR trigger treatment (open green triangle), and dual pro-TR and pro-CT hybrids with native TR trigger treatment (open purple diamond). Data are presented as the mean ± standard deviation (SD) of three experiments. Mean differences between experimental groups were tested with an unpaired t test. Values were significantly different at the *p < 0.05 or n.s. = not significant.