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.