Probing the serpin structural-transition mechanism in ovalbumin mutant R339T by proteolytic-cleavage kinetics of the reactive-centre loop.
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 further 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 x 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.
Arii
Yasuhiro
Y
Division of Applied Life Sciences, The Graduate School of Agriculture, Kyoto University, Uji, Kyoto 611-0011, Japan.
Hirose
Masaaki
M
D000818:Animals; D001665:Binding Sites; D002645:Chickens; D066298:In Vitro Techniques; D007700:Kinetics; D008958:Models, Molecular; D010047:Ovalbumin / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D017354:Point Mutation; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015843:Serpins / Q000737:chemistry* / Q000378:metabolism*; D013381:Subtilisins / Q000378:metabolism
D011994:Recombinant Proteins; D015843:Serpins; D010047:Ovalbumin; D013381:Subtilisins