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Enzymatic properties of barley α-amylase 1 (AMY1) are altered as a result of amino acid substitutions at subsites −5/−6 (Cys95Ala/Thr) and +1/+2 (Met298Ala/Asn/Ser) as well as in the double mutants, Cys95Ala/Met298Ala/Asn/Ser. Cys95Ala shows 176% activity towards insoluble Blue Starch compared to wild-type AMY1, kcat of 142 and 211% towards amylose DP17 and 2-chloro-4-nitrophenyl β-d-maltoheptaoside (Cl-PNPG7), respectively, but fivefold to 20-fold higher Km. The Cys95Thr-AMY1 AMY2 isozyme mimic exhibits the intermediary behaviour of Cys95Ala and wild-type. Met298Ala/Asn/Ser have slightly higher to slightly lower activity for starch and amylose, whereas kcat and kcat/Km for Cl-PNPG7 are ≤ 30% and ≤ 10% of wild-type, respectively. The activity of Cys95Ala/Met298Ala/Asn/Ser is 100–180% towards starch, and the kcat/Km is 15–30%, and 0.4–1.1% towards amylose and Cl-PNPG7, respectively, emphasizing the strong impact of the Cys95Ala mutation on activity. The mutants therefore prefer the longer substrates and the specificity ratios of starch/Cl-PNPG7 and amylose/Cl-PNPG7 are 2.8- to 270-fold and 1.2- to 60-fold larger, respectively, than of wild-type. Bond cleavage analyses show that Cys95 and Met298 mutations weaken malto-oligosaccharide binding near subsites −5 and +2, respectively. In the crystal structure Met298 CE and SD (i.e., the side chain methyl group and sulfur atom) are near C(6) and O(6) of the rings of the inhibitor acarbose at subsites +1 and +2, respectively, and Met298 mutants prefer amylose for glycogen, which is hydrolysed with a slightly lower activity than by wild-type. Met298 AMY1 mutants and wild-type release glucose from the nonreducing end of the main-chain of 6′′′-maltotriosyl-maltohexaose thus covering subsites −1 to +5, while productive binding of unbranched substrate involves subsites −3 to +3. |
