Dimerization and reactivation of triosephosphate isomerase in reverse micelles.
The reactivation of the homodimeric enzyme triosephosphate isomerase (TPI) was studied in reverse micelles. The enzyme was denatured in conventional aqueous mixtures with guanidine hydrochloride and transferred to reverse micelles formed with cetyltrimethylammonium bromide, hexanol, n-octane and water. In the transfer step, denatured TPI monomers distributed in single micelles, and guanidine hydrochloride was diluted more than 100 times. Under optimal reactivation conditions, 100% of the enzyme activity could be recovered. The rate of appearance of the catalytic activity increased with the concentration of protein, which indicated that catalysis required the formation of the dimer. The rate of TPI reactivation also increased with increasing protein concentration in the system with denatured TPI covalently derivatized at the catalytic site with the substrate analogue 3-chloroacetol phosphate. Thus, reactivation could take place via the formation of dimers composed of an inactive and an active subunit. Reactivation critically depended on the amount of water in the reverse micelles. The plot of the extent of reactivation versus the amount of water (2.5-7.0%) was markedly sigmoidal. Less than 20% reactivation took place with water concentrations below 3.5%, due to the formation (in less than 30 s) of stable inactive structures. The results indicate that reverse micelles provide a useful system to probe the events involved in the transformation of unfolded monomers to polymeric enzymes.
Garza-Ramos
G
G
Instituto de Fisiología Celular, Universidad Nacional Autónoma de México.
Tuena de Gómez-Puyou
M
M
Gómez-Puyou
A
A
Gracy
R W
RW
D000818:Animals; D002384:Catalysis; D004789:Enzyme Activation; D004793:Enzyme Reactivators; D019791:Guanidine; D006146:Guanidines; D007700:Kinetics; D046911:Macromolecular Substances; D008823:Micelles; D011489:Protein Denaturation; D011817:Rabbits; D012997:Solvents; D014305:Triose-Phosphate Isomerase / Q000378:metabolism*; D014867:Water
D004793:Enzyme Reactivators; D006146:Guanidines; D046911:Macromolecular Substances; D008823:Micelles; D012997:Solvents; D014867:Water; D014305:Triose-Phosphate Isomerase; D019791:Guanidine