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J Mol Biol. 1996 Mar 8;256(4):793-802.

Thermodynamic stability effects of single peptide bond hydrolysis in protein inhibitors of serine proteinases.

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Institute of Biochemistry, University of Wroclaw, Wroclaw, Poland.


Kunitz type soybean trypsin inhibitor (STI) and basic pancreatic trypsin inhibitor (BPTI) were used as model proteins to measure the thermodynamic consequences of single peptide bond hydrolysis. For each inhibitor the reactive site cleaved form was prepared and, additionally, each inhibitor was selectively cleaved at a Met residue. Selective cleavage generally led to reducing of the T(den) value from 7 K up to 75 K. For STI cleaved at Met84 a slight stabilization (increase of T(den) by 1.0 K) was observed. In terms of deltaG(den), the difference between the most extreme cleavage effect was 11.44 kcal/mole, much larger than resulting from the theoretical effects of crosslinks. It was found that hydrolysis of a single peptide bond affects not only entropy, but also enthalpy and heat capacity parameters. Moreover, the sign of change is opposite for two inhibitors: deltaH(den) and deltaS(den) increase for both cleaved forms of STI, while they decrease for two nicked forms of BPTI. To understand the stability effects, a thermodynamic cycle analysis was applied based on comparison of stabilities of intact and cleaved protein with peptide bond hydrolysis equilibria in native and denatured states. The cycle revealed a good agreement of the theoretical effect of crosslink elimination with a free energy difference for hydrolysis of a single peptide bond in a denatured protein. It appears that hydrolysis constants for single peptide bonds in a native protein span over at least 20 orders of magnitude. They are very low for peptide bonds placed in alpha-helices and very high if cleavage reaction leads to a formation of a new secondary structure element.

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