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

Mori, Haruhide
Bak-Jensen, Kristian Sass
Gottschalk, Tine E.
Motawia, Mohammed Saddik
Damager, Iben
Møller, Birger Lindberg
Svensson, Birte

¹Carlsberg Laboratory, Department of Chemistry, Gamle Carlsberg, Copenhagen Valby, Denmark
²Plant Biochemistry Laboratory, Department of Plant Biology, The Royal Veterinary and Agricultural University, Frederiksberg, Denmark

Correspondence to B. Svensson, Department of Chemistry, Carlsberg Laboratory, Gamle Carlsberg Vej10, DK-2500 Copenhagen Valby, Denmark. Fax: + 45 33 27 47 08, Tel.: + 45 33 27 53 45, E-mail: [email protected]

Abbreviations: AMY1, barley α-amylase AMY2, barley α-amylase 2; Cl-PNPG₇, 2-chloro-4-nitrophenyl β-D-maltoheptaoside; PNP, 4-nitrophenol; PNPG, 4-nitrophenyl α-D-glucoside; PNPG2, 4-nitrophenyl α-D-maltoside; PNPG3,4-nitrophenyl α-D-maltotrioside; PNPG4, 4-nitrophenyl α-D-maltotetraoside; PNPG5, 4-nitrophenyl α-D-maltopentaoside; PNPG6, 4-nitrophenyl α-D-maltohexaoside; PNPG7, 4-nitrophenyl α]-D-maltoheptaoside; IEF, isoelectric focusing.

Enzymes: α-amylase (α-glucan glucanohydrolase, EC 3.2.1.1); β-amylase (α-glucan maltohydrolase, EC 3.2.1.2); limit dextrinase (dextrin α-1,6-glucanohydrolase, EC 3.2.1.41).

^*Present address: Division of Applied Bioscience, Graduate School of Agriculture, Hokkaido University, Sapporo, Japan.

^†Present address: Pharmexa, Kogle Allé, Hørsholm, Denmark.

(Received 31 May 2001, revised 12 September 2001, accepted 19 October 2001)

European Journal of Biochemistry 268(24):p 6545-6558, December 2001.

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, k_cat of 142 and 211% towards amylose DP17 and 2-chloro-4-nitrophenyl β-D-maltoheptaoside (Cl-PNPG₇), 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-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 k_cat/K_m 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.