Dynamic Mechanism for the Serpin Loop Insertion as Revealed by Quantitative Kinetics

The serpin conformational change by insertion of the reactive center loop into β-sheet A plays a central role in multiple physiological consequences such as serine proteinase inhibition, latency and serpinopathic polymerization. To study the dynamic mechanism for the loop insertion, a novel kinetic method was established utilizing the ovalbumin mutant R339T/A352R; the loop insertion progressed after the cleavage of P1-P1′ (Arg352-Ser353) by trypsin was quenched at pH 8 and 0.5 °C, and different conformers were quantified by separation using ion-exchange HPLC. The apparent first-order rate constant kapp determined for various R339T/A352R derivatives differing in conformational stability was greatly increased by lowering the pH. The pH-dependence of kapp indicated that the protonation of side-chain(s) with a pKa value of around 4.6 is a pre-requisite for the loop insertion. The theoretical rate constant k for the protonated form calculated from kapp was highly variable, depending on the ovalbumin derivative; structural modifications that give increased mobility to helix F and the sheet-A half (s3A/s2A/s1A) resulted in a striking increase in the loop insertion rate constant k. The k values were determined at different temperatures for all the ovalbumin derivatives, and ΔH‡ and ΔS‡ values for the loop insertion reaction were determined according to the transition theory. The formation of the transition state was highly endothermic with minor entropy gain, requiring a ΔG‡ larger than 18 kcal/mol, which can offset the hydrogen-bond cleavages between s3A and s5A. These results are consistent with the transition state with an opened sheet A and altered orientation of helix F.