Abstract
Structural and mechanistic insights were revealed for the reversible inhibition of Porcine Pancreatic Elastase (PPE); the kinetics of uninhibited and inhibited hydrolysis of substrate Suc-AAA-pNA was analyzed thoroughly. Additionally, the interactions between PPE and its inhibitor were studied by computational techniques. The uninhibited hydrolysis of Suc-AAA-pNA by PPE proceeds through a virtual transition state, involving an inferior physical and another dominating chemical step, where two stabilized reactant states precede the predominant acyl-enzyme. Different kinds of bonding with the PPE-backbone residues, including those of the catalytic triad, were found during the MD simulation of 5 ns, as key interactions favoring a higher stabilization of the best ranked complex PPE-CF3C(O)-KA-NHPh-p-CF3. The proton inventories of the inhibited hydrolysis of Suc-AAA-pNA by PPE, were ruled out the existence of any virtual transition state and thus they argue for a different mode of catalysis involving a structurally disturbed PPE molecule. Thereafter, a novel inhibition mechanism was suggested.
Highlights
Elastases are proteolytic enzymes, which mainly hydrolyze proteins of the connective tissue in lungs, arteries, skin and ligaments[1,2]; they predominate in the pathogenesis of emphysema in the absence of suitable inhibitors[3]
The proton inventories of the inhibited hydrolysis of Suc-AAA-pNA by Porcine Pancreatic Elastase (PPE), were ruled out the existence of any virtual transition state and they argue for a different mode of catalysis involving a structurally disturbed PPE molecule
The objective of this work ensues inductively from the aforementioned results, whose the most decisive points are: In the absence of competitive inhibitors, the PPE initiates the catalytic hydrolysis of Suc-AAA-pNA by means of a virtual transition state, which is due to conformational changes toward the complex ES, and comprises two steps an inferior physical and another dominating chemical one
Summary
Elastases are proteolytic enzymes, which mainly hydrolyze proteins of the connective tissue in lungs, arteries, skin and ligaments[1,2]; they predominate in the pathogenesis of emphysema in the absence of suitable inhibitors[3]. The molecule of Porcine Pancreatic Elastase (PPE) comprises a polypeptide chain of 240 residues[4], whereas the occupancy of its S5 and S4 subsites, as well as the occurrence of proline and valine at P2 and P1, respectively, in its substrates is important for effective catalysis. Analysis of cocrystals of 1:1 complex of PPE and the reversible inhibitor CF3C(O)-LA-NHPh-p-CF3 (Ki 1⁄4 23.6 Â 10À 9 M), at 1.8 Aeffective resolution, showed the inhibitor molecule to be tightly bound on the PPE, with its trifluoromethylamide group to be bonded to the PPE active site, whereas both of its two fluorinated groups exhibited different specificities[5]
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