Kinetic monitoring of site-directed mutational β-amylase catalysis on a 27-MHz QCM

A quartz-crystal microbalance (QCM) technique was applied to analyze the effects of site-directed mutagenesis of soybean β-amylase (SBA) by using an amylose-immobilized 27-MHz QCM in buffer solution. We followed directly and quantitatively the formation and decomposition of an enzyme-substrate (ES) complex as frequency changes (mass changes) on the QCM plate. It has been predicted from X-ray crystallography of SBA that two carboxyl groups, Glu186 and Glu380, are the crucial residues for the catalytic hydrolysis of α-1,4-glycoside linkage, and the side chain Thr342 on the inner loop structure bends the glycoside at subsites −1 and +1 of the substrate, thereby accelerating the hydrolysis. When SBA wild-type (WT) was employed, the frequency increased (mass decreased) simply because of the hydrolysis of the substrate on the QCM plate. When T342S and T342A mutants having small side chains with no hydrogen bond on the inner loop were employed, the frequency change showed a sigmoidal curve pointing to an increase in ES complex formation and hydrolysis of the substrate. When E186A and E380A mutants were employed, the frequency decreases (mass increases) showed only enzyme binding to the substrate ES complex formation, but not hydrolysis. From curve fitting of these different patterns of frequency changes, we obtained all kinetic parameters for each step of the enzyme binding to the substrate (kon), the enzyme release from the ES complex (koff), and the catalytic rate constant (kcat) for WT and mutants. From the frequency change patterns of the substrate-immobilized QCM we evaluated how mutations of the enzyme affect kon, koff, and kcat values for ES complex formation and decomposition. We also evaluated the effects of accelerators and inhibitors on enzymes from the frequency change pattern.

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► A QCM was applied for site-directed mutagenesis of soybean β-amylase (SBA).
► Effects of mutations appeared on the time-course of each frequency change.
► Kinetic parameters of kon, koff, and kcat for mutants were obtained.