Dynamic mechanism for the serpin loop insertion as revealed by quantitative kinetics.
The serpin conformational change by insertion of the reactive center loop into beta-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 degrees C, and different conformers were quantified by separation using ion-exchange HPLC. The apparent first-order rate constant k(app) determined for various R339T/A352R derivatives differing in conformational stability was greatly increased by lowering the pH. The pH-dependence of k(app) indicated that the protonation of side-chain(s) with a pK(a) value of around 4.6 is a pre-requisite for the loop insertion. The theoretical rate constant k for the protonated form calculated from k(app) 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 DeltaH(double dagger) and DeltaS(double dagger) 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 DeltaG(double dagger) 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.
Takahashi
Nobuyuki
N
Division of Applied Life Sciences, The Graduate School of Agriculture, Kyoto University, Uji, Kyoto 611-0011, Japan.
Terakado
Kanako
K
Nakamura
Genichi
G
Soekmadji
Carolina
C
Masuoka
Toshio
T
Yamasaki
Masayuki
M
Hirose
Masaaki
M
D000818:Animals; D002645:Chickens; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009154:Mutation / Q000235:genetics; D010047:Ovalbumin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D011489:Protein Denaturation; D015843:Serpins / Q000378:metabolism*; D020860:Subtilisin / Q000378:metabolism; D013696:Temperature; D013816:Thermodynamics
D015843:Serpins; D010047:Ovalbumin; D020860:Subtilisin