ISSN:
0006-3592
Keywords:
Chemistry
;
Biochemistry and Biotechnology
Source:
Wiley InterScience Backfile Collection 1832-2000
Topics:
Biology
,
Process Engineering, Biotechnology, Nutrition Technology
Notes:
α-Galactosidase from soybean (Glycine max) was purified by a five-step procedure. The enzyme's natural substrates, raffinose and stachyose, have Km's of 3. 0 mM and 4. 79 mM, respectively. The products, galactose and sucrose, were measured after separation by liquid chromatography. Galactose is a competitive product inhibitor of stachyose and raffinose hydrolysis with a Ki of 0. 12 mM. We determined these parameters by an integral kinetic approach. Stachyose hydrolysis gives a nearly constant level of raffinose shortly after hydrolysis begins. Thus, cleavage of the first α-(1,6)-bond in the tetrasaccharide is the rate-limiting step. Since the stachyose hydrolysis yields raffinose, soybean α-galactosidase simultaneously hydrolyzes two substrates. We present a novel approach for analyzing simultaneous substrate hydrolysis with competitive product inhibition by a modified integral rate expression. The experimentally found kinetic parameters are confirmed by solving the simultaneous equations which describe the hydrolysis. This technique may be applicable to other hydrolytic enzymes with multiple substrates.
Additional Material:
9 Ill.
Type of Medium:
Electronic Resource
URL:
http://dx.doi.org/10.1002/bit.260350104
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