Modulation of activity and substrate binding modes by mutation of single and double subsites+1/+2 and-5/-6 of barley α-amylase 1

Enzymatic properties of barley alpha -amylase 1 (AMY1) are altered as a result of amino acid substitutions at subsites -5/-6 (Cys95 --> Ala/Thr) and +1/+2 (Met298 --> Ala/Asn/Ser) as well as in the double mutants, Cys95 --> Ala/Met298 --> Ala/Asn/Ser. Cys95 --> Ala shows 176% activity towards insoluble Blue Starch compared to wild-type AMY1, k(cat) of 142 and 211% towards amylose DP17 and 2-chloro-4-nitrophenyl beta -d-maltoheptaoside (Cl-PNPG(7)), respectively, but fivefold to 20-fold higher K-m. The Cys95 --> Thr-AMY1 AMY2 isozyme mimic exhibits the intermediary behaviour of Cys95 --> Ala and wild-type. Met298 --> Ala/Asn/Ser have slightly higher to slightly lower activity for starch and amylose, whereas k(cat) and k(cat)/K-m for Cl-PNPG(7) are less than or equal to 30% and less than or equal to 10% of wild-type, respectively. The activity of Cys95 --> Ala/Met298 --> Ala/Asn/Ser is 100-180% towards starch, and the k(cat)/K-m is 15-30%, and 0.4-1.1% towards amylose and Cl-PNPG(7), respectively, emphasizing the strong impact of the Cys95 --> Ala mutation on activity. The mutants therefore prefer the longer substrates and the specificity ratios of starch/Cl-PNPG(7) and amylose/Cl-PNPG(7) 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.