保守组氨酸残基在木糖氧化产碱杆菌木糖氧化亚种A-6的D-氨基酸酰化酶中的作用

Role of conserved histidine residues in D-aminoacylase from Alcaligenes xylosoxydans subsp. xylosoxydans A-6.

作者信息

Wakayama M, Yada H, Kanda S, Hayashi S, Yatsuda Y, Sakai K, Moriguchi M

机构信息

Department of Applied Chemistry, Faculty of Engineering, Oita University, Japan.

出版信息

Biosci Biotechnol Biochem. 2000 Jan;64(1):1-8. doi: 10.1271/bbb.64.1.

DOI:10.1271/bbb.64.1

PMID:10705441

Abstract

D-Aminoacylase from Alcaligenes xylosoxydans subsp. xylosoxydans A-6 (Alcaligenes A-6) was strongly inactivated by diethylpyrocarbonate (DEPC). An H67N mutant was barely active, with a kcat/Km 6.3 x 10(4) times lower than that of the recombinant wild-type enzyme, while the H67I mutant lost detectable activity. The H67N mutant had almost constant Km, but greatly decreased kcat. These results suggested that His67 is essential to the catalytic event. Both H69N and H69I mutants were overproduced in the insoluble fraction. The kcat/Km of H250N mutant was reduced by a factor of 2.5 x 10(4)-fold as compared with the wild-type enzyme. No significant difference between H251N mutant and wild-type enzymes in the Km and kcat was found. The Zn content of H250N mutant was nearly half of that of wild-type enzyme. These results suggest that the His250 residue might be essential to catalysis via Zn binding.

摘要

来自木糖氧化产碱杆菌木糖氧化亚种A-6（产碱杆菌A-6）的D-氨基酰化酶被焦碳酸二乙酯（DEPC）强烈灭活。H67N突变体几乎没有活性，其催化常数与米氏常数的比值（kcat/Km）比重组野生型酶低6.3×10⁴倍，而H67I突变体则失去了可检测到的活性。H67N突变体的米氏常数（Km）几乎不变，但催化常数（kcat）大幅降低。这些结果表明，组氨酸67对催化过程至关重要。H69N和H69I突变体均在不溶性部分中过量表达。与野生型酶相比，H250N突变体的kcat/Km降低了2.5×10⁴倍。未发现H251N突变体与野生型酶在Km和kcat上有显著差异。H250N突变体的锌含量几乎是野生型酶的一半。这些结果表明，组氨酸250残基可能通过与锌结合对催化作用至关重要。