Domain B protruding at the third β strand of the α/β barrel in barley α‐amylase confers distinct isozyme‐specific properties

Abstract

α‐Amylases belong to the α/β‐barrel protein family in which the active site is created by residues located at the C‐terminus of the β strands and in the helix‐connecting loops extending from these ends. In the α‐amylase family, a small separate domain B protrudes at the C‐terminus of the third β strand of the (β/α)8‐barrel framework. The 80% identical barley α‐amylase isozymes 1 and 2 (AMY1 and AMY2, respectively) differ in substrate affinity and turnover rate, CaCl2 stimulation of activity sensitivity to the endogenous 21‐kDa α‐amylase/subtilisin inhibitor, and stability at low pH. To identify regions that confer these isozyme‐specific variations, AMY1–AMY2 hybrid cDNAs were generated by in vivo homologous recombination in yeast. The hybrids AMY1‐(1‐90)–AMY2‐(90‐403) and AMY1‐(1‐161)–AMY2‐(161‐403) characterized in this study contain the 90‐residue and 161‐residue N‐terminal sequences, respectively, of AMY1 and complementary C‐terminal regions of AMY2. AMY1‐(1‐90)–AMY2‐(90‐403) comprises the 60‐amino‐acid domain B of AMY2 and resembles this isozyme in sensitivity to α‐amylase/subtilisin inhibitor and its low affinity for the substrates p‐nitrophenyl α‐d‐maltoheptaoside, amylose and the inhibitor acarbose. Only AMY1‐(1‐161)–AMY2‐(161‐403) and AMY1, which both share domain B, are stable at low pH. However, AMY2 and both hybrid AMY species, but not AMY1, show maximum enzyme activity on insoluble blue starch at approximately 10 mM CaCl2. Domain B thus determines several functional and stability properties that distinguish the barley α‐amylase isozymes. Copyright © 1994, Wiley Blackwell. All rights reserved