Factors that control the reactivity of the interface cysteine of triosephosphate isomerase from Trypanosoma brucei and Trypanosoma cruzi

The amino acid sequences and X-ray structures of homodimeric triosephosphat e isomerase from the pathogenic parasites Trypanosoma brucei (TbTIM) and Tr ypanosoma cruzi (TcTIM) are markedly similar. In the two TIMs, the side cha in of the only interface cysteine (Cys14) of one subunit docks into loop 3 of the other subunit. This portion of the interface is also markedly simila r in the two enzymes. Nonetheless, Cys14 of TcTIM is nearly 2 orders of mag nitude more susceptible to the thiol reagent methylmethane thiosulfonate (M MTS) than Cys14 of TbTIM, The causes of this difference were explored by me asuring the second-order rate constant of inactivation by MMTS (k(2)) under various conditions. At pH 7.4, k(2) in TcTIM is 70 times higher than in Tb TIM. The difference decreases to 30 when the amino acid sequence of loop 3 and adjoining residues of TbTIM are conferred to TcTIM (triple mutant). The pK(a) values of the thiol group of the interface cysteine of TcTIM and the triple mutant were 0.7 pH unit lower than in TbTIM. Because this differenc e could account for the different sensitivity of the enzymes to thiol reage nts, we determined the k(2) of inactivation at equal levels of ionization o f their interface cysteines. Under these conditions, the difference in k(2) between TcTIM and TbTIM became 8-fold, whereas that of the triple mutant t o TbTIM was 1.5 times. The substrate analogue phosphoglycolate did not modi fy the pK(a) of the thiol group of the interface, albeit it diminished the rate of its derivatization by MMTS. In the presence of phosphoglycolate, un der conditions in which the interface cysteines of the enzymes had equal le vels of protonation, the difference in k(2) of TcTIM and TbTIM became small er, whereas k(2) of the triple mutant was almost equal to that of TbTIM, Th us, from measurements of the reactivity of the interface cysteine in variou s conditions, it was possible to obtain information on the factors that con trol the dynamics of a portion of the dimer interface.