Characterization of the acidic and basic limbs of a bell-shaped pH profile in the inhibitory activity of bromelain inhibitor VI.
Bromelain inhibitor VI (BI-VI) is a cysteine proteinase inhibitor from pineapple stem and a unique two-chain inhibitor composed of two distinct domains. BI-VI's inhibitory activity toward the target enzyme bromelain is maximal at pH 4 and shows a bell-shaped pH profile with pKa values of about 2.5 and 5.3. This pH profile is quite different from that of bromelain, which is optimally active around pH 7. In the present article, to characterize the acidic limb, we first expressed the recombinant inhibitors designed to lose two putative hydrogen bonds of Ser7(NH)-Asp28(beta-CO2H) and Lys38(NH)-Asp51(beta-CO2H) and confirmed the existence of the hydrogen bonds by two-dimensional nuclear magnetic resonance (NMR). Moreover, it was revealed that these hydrogen bonds are not the essential electrostatic factor and some ionizable groups would be responsible for the acidic limb in the pH-inhibition profile. On the other hand, to characterize the basic limb, we examined the pH-dependent inhibition using the cysteine proteinase papain, some of whose properties differ from those of bromelain, and compared the data with the corresponding data for bromelain. The result suggests that the basic limb would be affected by some electrostatic factors, probably some carboxyl groups in the target proteinase.
Hatano
Ken-ichi
K
Department of Biological Sciences, Faculty of Engineering, Gunma University, 1-5-1 Tenjin-cho, Kiryu, Gunma 376-8515, Japan. hatano@bce.gunma-u.ac.jp
Sawano
Yoriko
Y
Miyakawa
Takuya
T
Tanokura
Masaru
M
D031230:Ananas / Q000201:enzymology; D001963:Bromelains / Q000037:antagonists & inhibitors*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009154:Mutation; D010206:Papain / Q000037:antagonists & inhibitors*; D010940:Plant Proteins / Q000737:chemistry* / Q000235:genetics; D018547:Plant Stems / Q000201:enzymology; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics
C095959:BI-VI protein, Ananas comosus; D010940:Plant Proteins; D011994:Recombinant Proteins; D001963:Bromelains; D010206:Papain