• BMB Reports
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• 제31권1호
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• pp.25-30
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• 1998

Kinetic studies of L-Alanine dehydrogenase from Bacillus subtilis-catalyzed reactions in the presence of $Zn^{2+}$ were carried out. The substrate (L-alanine) saturation curve is hyperbolic in the absence of the metal ion but it becomes sigmoidal when $Zn^{2+}$ is added to the reaction mixture indicating the positive cooperative binding of the substrate in the presence of zinc ion. The cooperativity of substrate binding depends on the xinc ion concentration: the Hill coefficients ($n_H$) varied from 1.0 to 1.95 when the zinc ion concentration varied from 0 to $60\;{\mu}m$. The inhibition of AlaDH by $Zn^{2+}$ is reversible and noncompetitive with respect to $NAD^+$ ($K_i\;=\;5.28{\times}10^{-5}\;M$). $Zn^{2+}$ itself binds to AlaDH with positive cooperativity and the cooperativity is independent of substrate concentration. The Hill coefficients of substrate biding in the presence of $Zn^{2+}$ are not affected by the enzyme concentration indicating that $Zn^{2+}$ binding does not change the polymerization-depolymerization equilibria of the enzyme. Among other metal ions, $Zn^{2+}$ appears to be a specific reversible inhibitor inducing conformational change through the intersubunit interaction. These results indicate that $Zn^{2+}$ is an allosteric competitive inhibitor and substrate being a non-cooperative per se, excludes the $Zn^{2+}$ from its binding site and thus exhibits positive cooperativity. The allosteric mechanism of AlaDh from Bacillus subtilis is consistent with both MWC and Koshland's allosteric model.

• 한국식품위생안전성학회지
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• 제15권1호
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• pp.51-54
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• 2000

1. E.coli에서 별도의 expression vector를 사용하지 않고도 Bacillus 균주 유래의 유용효소(D-Amino acid aminotransferase;D-AAT, Aspartate aminotransferase;AspAT, Alanine dehydrogenase;AlaDH 등)의 유전자의 5'-up stream부위의 발현 기구를 검토한 결과 각 유용 효소의 유전자들의 5'-upstream부위에 존재하는 프로모터들이 연속적으로 존재한다는 사실이 유추되었고, E.coli의 SD sequence와 매우 상동성이 높은 서열 또한 존재함을 확인하였다. 2. 유용 효소 유전자의 번역과 관련된 5'-upstream 부위의 분석을 통하여 유용 효소의 유전자들은 E.coli의 ribosomal RNA와 매우 안정한 SD pairing을 형성(D-AAT의 경우: -13.0kcal/mol, AspAT의 경우; -9.5kcal/mol, AlaDH의 경우 -15.8kca1/mol)할 수 있음을 확인하여, 이러한 높은 자유에너지 변화는 E.coli내에서 유용 효소의 번역에 기여함을 예상할 수 있다.