Effect of Modification of Sulfhydryl Groups in Soybean β -Amylase on the Interaction with Substrate and Inhibitors

Abstract

Methyl 2, 4-dinitrophenyl disulfide (MDPS) is shown to be an effective methanethiolating reagent for sulfhydryl groups in proteins via thiol-disulfide exchange reaction. It reacts with the two reactive sulfhydryl groups (SH1 and SH2) in soybean Β -amylase. A decrease of the enzymatic activity accompanies the methanethiolation of SH2. After complete methanethiolation of SH2, the modified enzyme still has 9% of the initial activity. Modification of SH2 with cyanide and iodoacetamide reduces the enzymatic activity to 65 and 2% of the initial activity, respectively. Apparently, the residual activity depends upon the size of the substituent at SH2. The modified enzymes still have the almost same K_m values for amylopectin and K_d values for enzyme-maltose and enzyme-cyclohexaamylose complexes as the native enzyme. In contrast to maltose and cyclohexaamylose, the K_d value of the enzymeglucose complex increases in the order of cyanide-, MDPS-, and iodoacetamide-modified enzymes, indicating that SH2 is located near the binding site of glucose. It is proposed from the subsite structure of soybean β -amylase that the position of SH2 and the glucose binding site is around subsite 1, where the nonreducing ends of the substrate bind productively.