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

• Published in: European journal of biochemistry Vol. 268; no. 24; pp. 6545 - 6558
• Main Authors: Mori, Haruhide, Sass Bak‐Jensen, Kristian, Gottschalk, Tine E., Saddik Motawia, Mohammed, Damager, Iben, Lindberg Møller, Birger, Svensson, Birte
• Format: Journal Article
• Language: English
• Published: Oxford, UK Blackwell Science Ltd 01.12.2001
• Subjects: (β/α)8 barrel; alpha-Amylases - chemistry; alpha-Amylases - genetics; alpha-Amylases - isolation & purification; alpha-Amylases - metabolism; Amylose - metabolism; Base Sequence; branched malto‐oligosaccharide; DNA Primers; Glycogen - metabolism; glycoside hydrolase family 13; Hordeum - enzymology; Hydrolysis; Models, Molecular; Mutagenesis, Site-Directed; Oligosaccharides - metabolism; Protein Binding; site‐directed mutagenesis; Substrate Specificity
• ISSN: 0014-2956; 1432-1033
• DOI: 10.1046/j.0014-2956.2001.02609.x

Enzymatic properties of barley α‐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, 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 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 kcat and kcat/Km for Cl‐PNPG7 are ≤ 30% and ≤ 10% of wild‐type, respectively. The activity of Cys95→Ala/Met298→Ala/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 Cys95→Ala 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.