A mutant ovalbumin (R339T), but not the wild-type protein, is transformed into the canonical loop-inserted, thermostabilized form after the P1—P1′ cleavage [Yamasaki, Arii, Mikami and Hirose (2002) J. Mol. Biol. 315, 113–120]. The loop-insertion mechanism in the ovalbumin mutant was investigated by proteolytic-cleavage kinetics. The nature of the inserted loop prevented futher cleavage of the P1—P1′ pre-cleaved R339T mutant by subtilisin, which cleaved the second P8—P7 loop site in the P1—P1′ pre-cleaved wild-type protein. After subtilisin proteolysis of the intact R339T, however, two final products that corresponded to the single P1—P1′ and double P1—P1′/P8—P7 cleavages were generated with variable ratios depending on the proteolysis conditions. This was accounted for by the occurrence of two mutually competitive reactions: the loop-insertion reaction and the proteolytic cleavage of the second P8—P7 site in the immediate intermediate after the P1—P1′ cleavage. The competitive nature of the two reactions enabled us to establish a kinetic method to determine the rate constants of the reactions. The first-order rate constant for the loop insertion was determined to be 4.0×10−3/s in the R339T mutant. The second-order rate constant for the P8—P7 cleavage in the immediate P1—P1′ cleavage product for the R339T mutant was >10 times compared with that for its wild-type counterpart. This highly accessible loop nature may play a crucial role in the loop-insertion mechanism for R339T mutant ovalbumin.
Probing the serpin structural-transition mechanism in ovalbumin mutant R339T by proteolytic-cleavage kinetics of the reactive-centre loop