作　　者： ;

机构地区： 西南大学

出　　处： 《BBA -蛋白质与蛋白酶学》

摘　　要： 从 Megalobatrachus 的碱的磷酸酶日本我们被 diethyl 焦性没食子酸碳酸盐(DEP ) 使失去活性。在里面激活在 pH 与 176 M ^?1 min ^?1 的一个秒顺序率常数跟随了伪 first-order 动力学 6.2 和 25 ° C。酶活动的损失在 242 nm 伴有吸收度的增加，使失去活性的酶被氢氧根胺重新激活，显示 histidine 残余的修正。这个结论被 pH 侧面也证实在激活，它与 pK _{6.6 的} 。甘油 3 磷酸盐，安培和磷酸盐的存在保护了酶免于在激活。结果表明 DEP 修改的 histidine 残余在活跃地点被定位。修改残余的 Spectrophotometric 数量化离子每在激活完全的导致的活跃地点显示出二 histidine 残余的那修正，但是运动 stoichiometry 显示修饰词的那一个分子与一个活跃地点反应了在期间在激活，而单个 histidine 残余的修正每活跃地点是足够的导致，可能建议二必要 histidine 残余为完全的活动每活跃地点是必要的在激活。 Alkaline phosphatase from Megalobatrachus japonicus was inactivated by diethyl pyrocarbonate (DEP). The inactivation followed pseudo-first-order kinetics with a second-order rate constant of 176 M-1 min(-1) at pH 6.2 and 25degreesC. The loss of enzyme activity was accompanied with an increase in absorbance at 242 nm and the inactivated enzyme was re-activated by hydroxylamine, indicating the modification of histidine residues. This conclusion was also confirmed by the PH profiles of inactivation, which showed the involvement of a residue with pK(a) of 6.6. The presence of glycerol 3-phosphate, AMP and phosphate protected the enzyme against inactivation. The results revealed that the histidine residues modified by DEP were located at the active site. Spectrophotometric quantification of modified residues showed that modification of two histidine residues per active site led to complete inactivation, but kinetic stoichiometry indicated that one molecule of modifier reacted with one active site during inactivation, probably suggesting that two essential histidine residues per active site are necessary for complete activity whereas modification of a single histidine residue per active site is enough to result in inactivation. (C) 2002 Elsevier Science B.V. All rights reserved.

关 键 词： 碱的磷酸酶 活跃地点 残余 化学修正 日本我们