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. 2003 Feb;10(2):115-9.
doi: 10.1038/nsb884.

Creation of a zymogen

Parit Plainkum  1 , Stephen M Fuchs, Suthep Wiyakrutta, Ronald T Raines
Affiliations

Creation of a zymogen

Parit Plainkum et al. Nat Struct Biol. 2003 Feb.

Abstract

Cells produce proteases as inactive zymogens. Here, we demonstrate that this tactic can extend beyond proteases. By linking the N and C termini of ribonuclease A, we obstruct the active site with the amino acid sequence recognized by plasmepsin II, a highly specific protease from Plasmodium falciparum. We generate new N and C termini by circular permutation. In the presence of plasmepsin II, a ribonuclease zymogen gains approximately 10(3)-fold in catalytic activity and maintains high conformational stability. We conclude that zymogen creation provides a new and versatile strategy for the control of enzymatic activity, as well as the potential development of chemotherapeutic agents.

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Conflict of interest statement

Competing interests statement

The authors declare that they have no competing financial interests.

Figures

Fig. 1
Fig. 1
Design of a ribonuclease zymogen. a, Scheme for creating a zymogen in which a circular permutation creates a steric block of the active site. b, Structural model of the unactivated ribonuclease A zymogen with 88/89 termini, 14-residue linker, and six disulfide bonds. The conformational energy of the non–wild-type residues was minimized with the program SYBYL. Atoms of the linker and cystines are shown explicitly, and the two nonnative cystines are labeled. c, Scheme of the primary sequence of ribonuclease A zymogens. The location of α-helices (cylinders) and β-strands (arrows) are indicated. The nine new termini, 14-residue linker, and four native and one nonnative (Cys4–Cys118) cystines are indicated.
Fig. 2
Fig. 2
Activation of ribonuclease A zymogen with 88/89 termini. Activation was monitored at different times after addition of plasmepsin II at a molar ratio of 1:100 (plasmepsin II:zymogen) by a, SDS–PAGE or b, ribonucleolytic activity. std, protein Mr standard; n, 0-min incubation without plasmepsin II; i0, 0-min incubation with unactivated plasmepsin II; i10, 10-min incubation with unactivated plasmepsin II.
Fig. 3
Fig. 3
Optimization of ribonuclease A zymogen. a, Effect of linker length of ribonucleolytic activity and conformational stability of ribonuclease A zymogen with 88/89 termini, before (open) and after (filled) activation by plasmepsin II. Zymogens have linkers of 13 (GS–KPIEFLELK–AG), 14 (GSG–KPIEFLELK–AG), or 15 (GSG–KPIEFLELK–GAG) residues. b, Effect of number of disulfide bonds on the ribonucleolytic activity and conformational stability of ribonuclease A zymogen with 88/89 termini, before (open) and after (filled) activation by plasmepsin II. Zymogens have four (native), five (native plus Cys4–Cys118), or six (native plus Cys4–Cys118 and Cys88–Cys89) disulfide bonds. c, Thermal unfolding of ribonuclease A zymogen with 88/89 termini and four native and two nonnative (Cys4–118 and Cys88–Cys89) disulfide bonds, before (left; Tm 50°C) and after (right; Tm 60°C) activation by plasmepsin II. Inserts: raw data from UV spectroscopy.
Fig. 4
Fig. 4
Scheme for the creation of plasmid that directs the expression of a ribonuclease A zymogen.
See this image and copyright information in PMC

References

    1. Von Der Helm K, Korant BD, Cheronis JC, editors. Proteases as Targets for Therapy. Springer–Verlag; Heidelberg, Germany: 2000.
    1. Smith J, Simons C. Proteinase and Peptidase Inhibition: Recent Potential Targets for Drug Development. Taylor & Francis; London, UK: 2002.
    1. Kühne W. Ueber die Verdauung der Eiweissstoffe durch den Pankreassaft. Virchows Arch. 1867;39:130–174.
    1. Khan AR, James MNG. Molecular mechanisms for the conversion of zymogens to active proteolytic enzymes. Protein Sci. 1998;7:815–836. - PMC - PubMed
    1. Laskowski M, Jr, Qasim MA, Lu SM. In: Protein–Protein Recognition. Kleanthous C, editor. Oxford University Press; Oxford, UK: 2000. pp. 228–279.

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