DEAMIDATION OF TRIOSEPHOSPHATE ISOMERASE IN REVERSE MICELLES - EFFECTS OF WATER ON CATALYSIS AND MOLECULAR WEAR AND TEAR

被引:13
作者
GARZARAMOS, G
DEGOMEZPUYOU, MT
GOMEZPUYOU, A
YUKSEL, KU
GRACY, RW
机构
[1] UNIV N TEXAS,HLTH SCI CTR,DEPT BIOCHEM & MOLEC BIOL,FT WORTH,TX 76107
[2] UNIV NACL AUTONOMA MEXICO,INST FISIOL CELULAR,MEXICO CITY,DF,MEXICO
关键词
D O I
10.1021/bi00188a027
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
The specific deamidation of asparagine-71 of triosephosphate isomerase increases upon substrate binding and catalysis. This deamidation at the dimer interface initiates subunit dissociation, unfolding, and protein degradation. The apparent connection between catalysis and terminal marking supports the concept of ''molecular wear and tear'', and raises questions related to the molecular events that lead to deamidation. In order to explore this interaction, triosephosphate isomerase was entrapped in reverse micelles with different water contents that support different catalytic rates. Deamidation was quantified for the free enzyme, the enzyme in the presence of substrates, and the enzyme which had been covalently modified at the catalytic center with the substrate analogue 3-chloroacetol phosphate (CAP). Both in water and in reverse micelles of cetyltrimethylammonium with 3% and 6% water, substrate binding enhanced deamidation. Studies of the extent of deamidation at various water concentrations showed that deamidation per catalytic turnover was about 6 and 17 times higher in 6% and 3% water than in 100% water, respectively. The enzyme was also entrapped in micelles formed with toluene, phospholipids, and Triton X-100 to explore the process at. much lower water concentrations (e.g., 0.3%). Under these conditions, catalysis was very low, and hardly any deamidation took place. Deamidation of the CAP-labeled enzyme was also markedly diminished. At these low-water conditions, the enzyme exhibited markedly increased thermostability and resistance to hydrolysis of the amide bonds. The data suggest that the rate of deamidation not only is dependent on the number of catalytic events but also is related to the time that asparagine-71 exists in a conformation or solvent environment more favorable for deamidation.
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页码:6960 / 6965
页数:6
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