大肠杆菌3-异丙基苹果酸脱氢酶的纯化、催化特性及热稳定性

Purification, catalytic properties and thermostability of 3-isopropylmalate dehydrogenase from Escherichia coli.

作者信息

Wallon G, Yamamoto K, Kirino H, Yamagishi A, Lovett S T, Petsko G A, Oshima T

机构信息

Rosenstiel Medical Sciences Research Center, Brandeis University, Waltham, MA 02254-9110, USA.

出版信息

Biochim Biophys Acta. 1997 Jan 4;1337(1):105-12. doi: 10.1016/s0167-4838(96)00157-4.

DOI:10.1016/s0167-4838(96)00157-4

PMID:9003442

Abstract

3-isopropylmalate dehydrogenase (IPMDH) from Escherichia coli was overexpressed, purified and crystallized. The enzyme was characterized and compared to its thermophilic counterpart from Thermus thermophilus strain HB8. As in the thermophile enzyme, the activity of E. coli IPMDH was dependent on the divalent cations, Mg2+ or Mn2+, with Mn2+ being the preferred cation. Activity was also strongly influenced by KCl: 0.3 M were necessary for the optimal activity. At 40 degrees C the K(m) of E. coli IPMDH was 105 microM for IPM and 321 microM for NAD, the kcat was 69 s-1. The half denaturation temperature was 64 degrees C, which was 20 degrees C lower than that of the thermophile enzyme.

摘要

来自大肠杆菌的3-异丙基苹果酸脱氢酶（IPMDH）被过量表达、纯化并结晶。对该酶进行了表征，并与嗜热栖热菌HB8菌株的嗜热对应物进行了比较。与嗜热菌酶一样，大肠杆菌IPMDH的活性依赖于二价阳离子Mg2+或Mn2+，其中Mn2+是首选阳离子。KCl也对活性有强烈影响：最佳活性需要0.3M。在40℃时，大肠杆菌IPMDH对IPM的Km为105μM，对NAD的Km为321μM，kcat为69 s-1。半变性温度为64℃，比嗜热菌酶低20℃。