STUDIES OF THE INTERACTION OF SOYBEAN TRYPSIN INHIBITOR (KUNITZ) (STI) WITH TRYPSIN AND CARBOXYPEPTIDASE B (CPB)

MARY WELCH BAILLARGEON, Purdue University

Abstract

The reactive site Arg('63)-Ile('64) peptide bond of soybean trypsin inhibitor (STI) is preferentially and reversibly hydrolyzed by trypsin. Carboxypeptidase B (CPB) specifically and reversibly hydrolyzes the new free carboxyl terminal, Arg('63). The interaction of STI and trypsin was studied by CPB catalyzed incorporation of ('13)C enriched arginine into STI. A modification of the method of Sealock and Laskowski {(1969) Biochemistry 8, 3703-3710} incorporating trypsin - Sepharose 4B was used to replace Arg('63) of STI with {('13)C('(zeta))}arginine and {('13)C(DEGREES)}arginine. Protein purity was established by disc gel electrophoresis. The ('13)C NMR spectrum of {{('13)C('(zeta))}Arg('63)}-STI revealed a single peak with chemical shift 157.81 ppm (downfield from (CH(,3))(,4)Si). In complex with bovine or porcine trypsin, the peak assigned to Arg('63) C('(zeta)) shifted in 158.04 ppm. The negligible chemical shift change indicates that there is no significant proton transfer from the guanidinum group upon complex formation. The ('13)C NMR spectrum of {{('13)C(DEGREES))Arg('63)}-STI revealed one peak at 173.8 ppm. When a half stoichiometric amount of bovine trypsin was added to labeled STI, two ('13)C NMR peaks were observed, at 173.8 ppm and 174.7 ppm. Disc gel electrophoresis identified the two species as {{('13)C(DEGREES))Arg('63)}-STI and the complex, {{('13)C(DEGREES)}Arg('63)}-STI-trypsin. After treatment with sufficient bovine trypsin to yield only a small excess of labeled STI, a single peak remained at 174.7 ppm. Model studies lead to a proposed chemical shift of 118 (+OR-) 10 ppm for a covalent, tetrahedral intermediate between STI and trypsin. The absence of a large upfield shift of the {('13)C(DEGREES)}Arg('63) peak upon complexation establishes that the STI-trypsin complex is not a covalent, fully tetrahedral adduct, as proposed for the STI-trypsin complex {Sweet, R. M., Wright, H. T., Janin, J., Chothia, C. H., and Blow, D. M. (1974) Biochemistry 13, 4212-4228}. The present ('13)C NMR results do not distinguish between a complex in which C(DEGREES) is completely trigonal and the structure recently proposed for PTI-trypsin based on X-ray studies, where C(DEGREES) is partially tetrahedral due to constraints imposed by the enzyme-inhibitor contact {Huber, R. and Bode, W. (1978) in "Federation of European Biochemical Societies, 11th Meeting Copenhagen 1977" (Magnussen, S., et al., Eds.) pp. 15-34, Pergamon Press, Elmsford, NY}. In labeled modified STI, {{('13)C(DEGREES)}Arg('63)}-STI(63,64), the chemical shift of the labeled carbon was pH dependent as expected (179.0 ppm at pH 7.0, 174.7 ppm at pH 2.5). The interaction of STI(63,64) and CPB was measured kinetically in both the hydrolytic and the synthetic directions in 0.05 M piperazine-N,N'-bis(2-ethanesulfonic acid), 0.14 M KCl, 0.03 M CaCl(,2), pH 6.7. The reaction remained first order under all experimental conditions used. For the hydrolytic reaction, (k(,cat)/K(,m))(,f) is 2000 s('-1) M ('-1); K(,m) and K(,iarg) are 1.1 x 10('-3) M. For the synthetic reaction, only (k(,cat)/K(,m))(,r) was measurable (0.028 s('-1) M('-1) in the presence of 0.1 M arginine). The rate data allow the determination of the equilibrium constant for the reaction in the hydrolytic direction to be calculated to be 10('2) M. The interaction of PTI(15,16) with CPB was also studied in the hydrolytic direction. The reaction was again first order. K(,m) for PTI(15,16) is the same as K(,m) for STI(63,64) within experimental error; k(,cat) is 12.3 s('-1). The kinetic results can be explained by an ordered Uni Bi mechanism in which arginine is released last. However, a rapid equilibrium random mechanism cannot be excluded.

Degree

Ph.D.

Subject Area

Biochemistry

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