tailieunhanh - Báo cáo Y học: Barley a-amylase Met53 situated at the high-affinity subsite )2 belongs to a substrate binding motif in the bfia loop 2 of the catalytic (b/a)8-barrel and is critical for activity and substrate specificity

Met53 in barleya-amylase 1 (AMY1) is situated at the high-affinity subsite)2. While Met53 is unique to planta-amy-lases,the adjacent Tyr52 stacks onto substrate at subsite)1 and is essentially invariant in glycoside hydrolase family 13. These residues belong toa short sequencemotif inbfialoop 2 of the catalytic (b/a)8-barrel and site-directedmutagenesis was used to introduce a representative variety of structural changes,Met53Glu/Ala/Ser/Gly/Asp/Tyr/Trp,to investi-gate the role of Met53. | Eur. J. Biochem. 269 .3 7 _v90 22002 FEBS 2002 doi Barley a-amylase Met53 situated at the high-affinity subsite -2 belongs to a substrate binding motif in the fia loop 2 of the catalytic p a 8-barrel and is critical for activity and substrate specificity Haruhide Mori Kristian Sass Bak-Jensen and Birte Svensson Carlsberg Laboratory Department of Chemistry Gamle Carlsberg Vej 10 Copenhagen Valby Denmark Met53 in barley a-amylase 1 AMY1 is situated at the high-affinity subsite -2. While Met53 is unique to plant a-amy-lases hie adjacent Tit52 slacks onto subtlrale al subsite -1 and is essentially invariant in glycoside hydrolase family 13. These residues belong to a short sequence motif in Pfia loop 2 of the catalytic P a 8-barrel and site-directed mutagenesis was used to introduce a representative variety of structural changes Met33Glu Ala Ser Gly Asp Tyr Trp tin ie sti-gate the role of Met53. Compared to wild-type Met53Ghu Asp AMY1 displayed 117 90 activity towards insoluble Blue Starch and M2t53A strUr Gly U2-U3-38 l but Met53Tyr Tyr only even ihoihi bodt Asp and rp occur frequently at this position in family 13. Towards amylose DP17 degree of polymerization 17 and 2-chloro-4-nitrophenyl P-D-maltoheptaoside the activity kc Kn of all mutants was reduced to and of wild-type yespeciv y . Km increased up to 20-fold for these soluble substrates and the attacl on glucosidic linkages in 4-nitrophenyl a-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. Met53Tyr affected the action pattern on PNPG7 ihiihi highly unusual