Barley α-amylase Met53 situated at the high-affinity subsite −2 belongs to a substrate binding motif in the β→α loop 2 of the catalytic (β/α)8-barrel and is critical for activity and substrate specificity

  • Mori, Haruhide
  • Bak-Jensen, Kristian Sass
  • Svensson, Birte

  • Carlsberg Laboratory, Department of Chemistry, Gamle Carlsberg Vej 10, Copenhagen Valby, Denmark

Correspondence to B. Svensson, Carlsberg Laboratory, Department of Chemistry, Gamle Carlsberg Vej 10, DK-2500 Copenhagen Valby, Denmark. Fax: + 45 33 27 47 08, Tel.: + 45 33 27 53 45, E-mail: [email protected]

*Present address: Division of Applied Bioscience, Graduate School of Agriculture, Hokkaido University, Sapporo 060-8589, Japan

Abbreviations: AMY1, barley α-amylase 1; AMY2, barley α-amylase 2; Cl-PNPG7, 2-chloro-4-nitrophenyl βD-maltoheptaoside; DP17, degree of polymerization = 17; GH13, glycoside hydrolase family 13; GH-H, clan H of glycoside hydrolases; PNPG, 4-nitrophenyl α-D-glucoside; PNPG2–PNPG12, 4-nitrophenyl α-D-maltoside through 4-nitrophenyl α-D-maltododecaoside; TAA, Taka-amylase A.

Enzyme: α-Amylase (α-1,4-D-glucan glucanohydrolase, EC 3.2.1.1).

(Received 12 April 2002, revised 28 June 2002, accepted 14 August 2002)

European Journal of Biochemistry 269(22):p 5377-5390, November 2002.

Met53 in barley α-amylase 1 (AMY1) is situated at the high-affinity subsite −2. While Met53 is unique to plant α-amylases, the adjacent Tyr52 stacks onto substrate at subsite −1 and is essentially invariant in glycoside hydrolase family 13. These residues belong to a short sequence motif in β→α loop 2 of the catalytic (β/α)8-barrel and site-directed mutagenesis was used to introduce a representative variety of structural changes, Met53Glu/Ala/Ser/Gly/Asp/Tyr/Trp, to investigate the role of Met53. Compared to wild-type, Met53Glu/Asp AMY1 displayed 117/90% activity towards insoluble Blue Starch, and Met53Ala/Ser/Gly 76/58/38%, but Met53Tyr/Trp only 0.9/0.1%, even though both Asp and Trp occur frequently at this position in family 13. Towards amylose DP17 (degree of polymerization = 17) and 2-chloro-4-nitrophenyl β-D-maltoheptaoside the activity (kcat/Km) of all mutants was reduced to 5.5–0.01 and 1.7–0.02% of wild-type, respectively. Km increased up to 20-fold for these soluble substrates and the attack on glucosidic linkages in 4-nitrophenyl α-D-maltohexaoside (PNPG6) and PNPG5 was determined by action pattern analysis to shift to be closer to the nonreducing end. This indicated that side chain replacement at subsite −2 weakened substrate glycon moiety contacts. Thus whereas all mutants produced mainly PNPG2 from PNPG6 and similar amounts of PNPG2 and PNPG3 accounting for 85% of the products from PNPG5, wild-type released 4-nitrophenol from PNPG6 and PNPG and PNPG2 in equal amounts from PNPG5. Met53Trp affected the action pattern on PNPG7, which was highly unusual for AMY1 subsite mutants. It was also the sole mutant to catalyze substantial transglycosylation – promoted probably by slow substrate hydrolysis – to produce up to maltoundecaose from PNPG6.

Copyright © 2002 Blackwell Science Ltd.