Kinetics of Interaction of Trypsin with an Anionic Inhibitor of Trypsin BWI-1a from Buckwheat Seeds

I. P. Gladysheva^1*, D. P. Gladyshev¹, Y. E. Dunaevsky², M. A. Belozersky², and N. I. Larionova¹

¹School of Chemistry, Lomonosov Moscow State University, Moscow, 119899 Russia; fax: (095) 939-5417; E-mail: LAR_LAB@ENZYME.CHEM.MSU.RU

²Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Moscow, 119899 Russia; fax: (095) 939-3181

^* To whom correspondence should be addressed.

Received December 22, 1998; Revision received February 19, 1999

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The kinetics of binding of bovine trypsin to a proteinaceous inhibitor of trypsin from buckwheat seeds (BWI-1a) has been studied. The association rate constant (k_ass) was 2.2·10⁶ M^-1·sec^-1 and the dissociation rate constant (k_off) of the enzyme--inhibitor complex was 3.5·10^-3 sec^-1; the inhibition constant K_i was 1.5 nM. The inhibitor BWI-1a is of the slow, tightly binding type. The mechanism of the inhibition of bovine trypsin by the trypsin inhibitor BWI-1a was studied. The mechanism of inhibition was found to involve two steps according to the kinetic data.

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KEY WORDS: proteinase inhibitor, buckwheat, kinetics, inhibition constant, mechanism

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