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Barley alpha-amylase isozymes 1 (AMY1) and 2 (AMY2) have 80% sequence identity but possess different physico-chemical properties. By incubation in the range 37–85 °C T50 is 75.2 °C of AMY1 and 79.2 °C of AMY2. While AMY2 is also most stable in urea at pH 6.7, [urea]50 being 8.2 M compared to 7.9 M for AMY1, AMY1 has highest stability in urea below pH 6 or in the presence of NaCl. Moreover AMY1 is most stable in guanidinium chloride. Charge screening thus destabilises AMY2 but stabilises AMY1. Isozyme sequence comparison suggests that AMY1 lacks four of the 20 salt-bridges identified in the crystal structure of AMY2. The four residues that differ comprise Lys67AMY2 and Asp267AMY2, forming salt-bridges on the surface of the catalytic (β/α)8-barrel (domain A), and Glu96AMY2 and His344AMY2 that participate in charged networks between domain A and the small domain B and the C-terminal domain, respectively. Four corresponding AMY2 mimics A68K; D97E; Q269D; N346H were made in AMY1 by site-directed mutagenesis. While D97E and Q269D have slightly improved stability compared to AMY1 wild-type, N346H and, under certain conditions, A68K are destabilised. The four mutants show 22–176% activity (kcat/Km) toward 2-chloro-4-nitrophenol β- d -maltoheptaoside and amylose DP17 and 43–117% activity for insoluble starch. |
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