Derivatization of the interface cysteine of triosephosphate isomerase fromTrypanosoma brucei and Trypanosoma cruzi as probe of the interrelationshipbetween the catalytic sites and the dimer interface

In the interface of homodimeric triosephosphate isomerase from Trypanosoma brucei (TbTIM) and Trypanosoma cruzi (TcTIM), one cysteine of each monomer forms part of the intersubunit contacts, The relatively slow derivatization of these cysteines by sulfhydryl reagents induces progressive structural a lterations and abolition of catalysis [Garza-Ramos et al. (1998) Eur. J. Bi ochem. 253, 684-691]. Derivatization of the interface cysteine by 5,5-dithi obis(2-nitrobenzoate) (DTNB) and methylmethane thiosulfonate (MMTS) was use d to probe if events at the catalytic site are transmitted to the dimer int erface. It was found that enzymes in the active catalytic state are signifi cantly less sensitive to the thiol reagents than in the resting state. Maxi mal protection against derivatization of the interface cysteine by thiol re agents was obtained at near-saturating substrate concentrations, Continuous recording of derivatization by DTNB showed that catalysis hinders the reac tion of sulfhydryl reagents with the interface cysteine. Therefore, in addi tion to intrinsic structural barriers, catalysis imposes additional impedim ents to the action of thiol reagents on the interface cysteine. In TcTIM, t he substrate analogue phosphoglycolate protected strongly against DTNB acti on, and to a lesser extent against MMTS action; in TbTIM, phosphoglycolate protected against the effect of DTNB, but not against the action of MMTS. T his indicates that barriers of different magnitude to the reaction of thiol reagents with the interface cysteine are induced by the events at the cata lytic site. Studies with a Cys14Ser mutant of TbTIM confirmed that all the described effects of sulfhydryl reagents on the trypanosomal enzymes are a consequence of derivatization of the interface cysteine.