title: Increasing the hydrolysis constant of the reactive site upon introduction of an engineered Cys¹⁴-Cys³⁹ bond into the ovomucoid third domain from silver pheasant.
abstract: P14C/N39C is the disulfide variant of the ovomucoid third domain from silver pheasant (OMSVP3) introducing an engineered Cys¹⁴-Cys³⁹ bond near the reactive site on the basis of the sequence homology between OMSVP3 and ascidian trypsin inhibitor. This variant exhibits a narrower inhibitory specificity. We have examined the effects of introducing a Cys¹⁴-Cys³⁹ bond into the flexible N-terminal loop of OMSVP3 on the thermodynamics of the reactive site peptide bond hydrolysis, as well as the thermal stability of reactive site intact inhibitors. P14C/N39C can be selectively cleaved by Streptomyces griseus protease B at the reactive site of OMSVP3 to form a reactive site modified inhibitor. The conversion rate of intact to modified P14C/N39C is much faster than that for wild type under any pH condition. The pH-independent hydrolysis constant (K(hyd) °) is estimated to be approximately 5.5 for P14C/N39C, which is higher than the value of 1.6 for natural OMSVP3. The reactive site modified form of P14C/N39C is thermodynamically more stable than the intact one. Thermal denaturation experiments using intact inhibitors show that the temperature at the midpoint of unfolding at pH 2.0 is 59 °C for P14C/N39C and 58 °C for wild type. There have been no examples, except P14C/N39C, where introducing an engineered disulfide causes a significant increase in K(hyd) °, but has no effect on the thermal stability. The site-specific disulfide introduction into the flexible N-terminal loop of natural Kazal-type inhibitors would be useful to further characterize the thermodynamics of the reactive site peptide bond hydrolysis.
authors.affiliation: National Food Research Institute, National Agriculture and Food Research Organization, Kannondai, Tsukuba, Ibaraki 305-8642, Japan.
authors.forename: Hikaru
authors.initials: H
authors.lastname: Hemmi
authors.forename: Takashi
authors.initials: T
authors.lastname: Kumazaki
authors.forename: Shuichi
authors.initials: S
authors.lastname: Kojima
authors.forename: Takuya
authors.initials: T
authors.lastname: Yoshida
authors.forename: Tadayasu
authors.initials: T
authors.lastname: Ohkubo
authors.forename: Hideyoshi
authors.initials: H
authors.lastname: Yokosawa
authors.forename: Kin-Ichiro
authors.initials: K
authors.lastname: Miura
authors.forename: Yuji
authors.initials: Y
authors.lastname: Kobayashi
mesh_terms: D000818:Animals; D001665:Binding Sites; D001717:Birds; D003545:Cysteine / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D008958:Models, Molecular; D010059:Ovomucin / Q000737:chemistry* / Q000494:pharmacology*; D013816:Thermodynamics; D014361:Trypsin Inhibitors / Q000737:chemistry* / Q000494:pharmacology*
chemical_list: D014361:Trypsin Inhibitors; D010059:Ovomucin; D003545:Cysteine