Interaction of Sweet Potato β-Amylase with Its Reaction Product, Maltose

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Abstract
To identify amino acid residues present in the active site of sweet potato β-amylase, the interaction of the reaction product of the enzyme, maltose, has been studied. Addition of maltose to β-amylase solution produced a difference spectrum, which indicates that four out of forty tryptophan residues in the tetrameric enzyme have specific interactions with maltose. The sugar also affects the chemical modification of sulfhydryl groups in β-amylase with p-mercuribenzoate (pMB). Modification of four out of ten sulfhydryl groups which are reactive in the native state is prevented almost completely and that of two is retarded by maltose. Inactivation of β-amylase is accompanied by the modification of the two slowly reactive residues. The interaction between maltose and the enzyme is further manifested by the formation of crystals of β-amylase from a buffer solution containing maltose. A method we have developed shows that the crystals are those of a complex of four maltoses with one enzyme molecule. These results strongly suggest that four maltoses bind to four substrate binding sites, in which four tryptophan and six cysteinyl residues are located. During the course of the study very unusual results were obtained, namely, restoration of β-amylase activity and release of mercury from β-amylase simply by dialysis of the enzyme in which ten sulfhydryl groups had been modified with pMB to produce an almost inactive protein.