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

The specific deamidation of asparagine-71 of triosephosphate isomerase increases upon substrate binding and catalysis. This deamidation at t he dimer interface initiates subunit dissociation, unfolding, and prot ein degradation. The apparent connection between catalysis and termina l marking supports the concept of ''molecular wear and tear'', and rai ses 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 suppo rt 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 substra te analogue 3-chloroacetol phosphate (CAP). Both in water and in rever se 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 tur nover 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 a t. much lower water concentrations (e.g., 0.3%). Under these condition s, catalysis was very low, and hardly any deamidation took place. Deam idation of the CAP-labeled enzyme was also markedly diminished. At the se low-water conditions, the enzyme exhibited markedly increased therm ostability and resistance to hydrolysis of the amide bonds. The data s uggest that the rate of deamidation not only is dependent on the numbe r of catalytic events but also is related to the time that asparagine- 71 exists in a conformation or solvent environment more favorable for deamidation.