Chemical composition and properties of soybean beta-amylase.
The molecular weight of soybean beta-amylase [EC 3.2.1.2] was determined to be 57,000 daltons by the sedimentation equilibrium method, and the enzyme was found to consist of 494 amino acid residues. No difference was found in molecular weight or composition between two components of beta-amylase separated by ion-exchange chromatography. The N-terminus of the enzyme was not detectable by the fluorodinitrobenzene method or phenylisothiocyanate method, and the C-terminus was determined to be alanine by the carboxypeptidase [EC 3.4.12.2] method. Five half-cystine residues were found in the form of cysteine; all the sulfhydryl groups could be titrated by p-chloromercuribenzoate after denaturation of the enzyme with guanidine hydrochloride, but only some in the native enzyme. The rates of mercaptide formation of these groups were dependent on pH and were different from each other, all being much lower than the rate for the free sulfhydryl group in mercaptoethanol. Differential titration experiments at different pH's and in the presence of maltose showed that mercaptide formation by only one sulfhydryl group caused loss of activity, and the reaction was accompanied by changes in the environment around aromatic side chains in the enzyme, which were detected by difference spectra and fluorescence emission spectra. These facts suggest that modification of the sulfhydryl groups causes a conformational change of the enzyme. Some preliminary crystallographic data for crystals formed at pH 4.0 were obtained, and inactivation by heavy metal salts was examined in relation to the preparation of isomorphous heavy atom derivatives.
D000596:Amino Acids / Q000032:analysis; D000681:Amylases / Q000378:metabolism*; D001665:Binding Sites; D002729:Chloromercuribenzoates / Q000494:pharmacology; D007700:Kinetics; D008320:Maltose / Q000494:pharmacology; D008623:Mercaptoethanol / Q000494:pharmacology; D008670:Metals / Q000494:pharmacology; D008970:Molecular Weight; D010944:Plants / Q000201:enzymology*; D013025:Glycine max; D013438:Sulfhydryl Compounds / Q000032:analysis; D014961:X-Ray Diffraction
D000596:Amino Acids; D002729:Chloromercuribenzoates; D008670:Metals; D013438:Sulfhydryl Compounds; D008623:Mercaptoethanol; D008320:Maltose; D000681:Amylases