Loosely packed papain prosegment displays inhibitory activity.
Most protease prosegments are co-synthesized at the N-termini of cysteine proteases and are involved in folding assistance, inhibition, and activation of their mature enzymes. By using circular dichroism, UV-difference and fluorescence spectroscopies, we studied the thermal unfolding of papain prosegment. The transition seems to be two-state and reversible, with an unfolded state prone to aggregation. Unfolding thermodynamic parameters obtained show low values both for deltaH(Tm) and deltaCp(U), indicative of a loosely packed three-dimensional conformation for the prosegment at near-neutral pH conditions. In spite of these results, fluorescence experiments demonstrate that papain prosegment is able to recognize and inhibit its cognate protease. An acid medium induces a molten globule-like state without intermediates, which in turn undergoes an irreversible thermal unfolding. Our results suggest that papain prosegment has a high degree of conformational flexibility, with the ability to form not only a molten globule-like structure in activating conditions, but also requiring an induced fit in order to be functional as inhibitor.
Gutiérrez-González
Luis H
LH
Area de Biofisicoquímica, Departamento de Química, Universidad Autónoma Metropolitana-Iztapalapa, Apartado Postal 55-534, 09340 Mexico City, Mexico.
Rojo-Domínguez
Arturo
A
Cabrera-González
Nallely E
NE
Pérez-Montfort
Ruy
R
Padilla-Zúñiga
A Jaqueline
AJ
D000818:Animals; D002942:Circular Dichroism; D004791:Enzyme Inhibitors / Q000737:chemistry; D005813:Genes, Synthetic / Q000235:genetics; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008958:Models, Molecular; D010206:Papain / Q000037:antagonists & inhibitors / Q000737:chemistry*; D011489:Protein Denaturation; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry; D013050:Spectrometry, Fluorescence; D013816:Thermodynamics
D004791:Enzyme Inhibitors; D011994:Recombinant Proteins; D010206:Papain