Increasing the hydrolysis constant of the reactive site upon introduction of an engineered Cys14Cys39 bond into the ovomucoid third domain from silver pheasant

• Published in: Journal of peptide science Vol. 17; no. 8; pp. 595 - 600
• Main Authors: Hemmi, Hikaru, Kumazaki, Takashi, Kojima, Shuichi, Yoshida, Takuya, Ohkubo, Tadayasu, Yokosawa, Hideyoshi, Miura, Kin-ichiro, Kobayashi, Yuji
• Format: Journal Article
• Language: English
• Published: Chichester, UK John Wiley & Sons, Ltd 01.08.2011
• Subjects: Animals; Binding Sites; Birds; circular dichroism; Cysteine - chemistry; engineered disulfide bond; Hydrogen-Ion Concentration; Hydrolysis; hydrolysis constant of the reactive site; inhibitory specificity; Models, Molecular; Ovomucin - chemistry; Ovomucin - pharmacology; serine protease inhibitors; thermal stability; Thermodynamics; Trypsin Inhibitors - chemistry; Trypsin Inhibitors - pharmacology
• ISSN: 1075-2617; 1099-1387
• DOI: 10.1002/psc.1381

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 (Khyd°) 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 Khyd°, 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. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd. P14C/N39C, the CSH‐motif‐introducing variant of OMSVP3, exhibits significant enhanced susceptibility toward a target protease. The conversion rate from intact to modified form of P14C/N39C is faster than that for wild type. Furthermore, P14C/N39C causes relatively large increase in the hydrolysis constant(Khyd°), while it has almost the same thermal stability as wild type. There have been no examples except P14C/N39C where an engineered disulfide introduction could cause a significant increase in Khyd° without affecting any effect on the thermal stability.