The amino acid sequences and X-ray structures of homodimeric triosephosphate isomerase from the pathogenic parasites Trypanosoma brucei (TbTIM) and Trypanosoma cruzi (TcTIM) are markedly similar. In the two TIMs, the side chain 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 similar in the two enzymes. Nonetheless, Cys14 of TcTIM is nearly 2 orders of magnitude more susceptible to the thiol reagent methylmethane thiosulfonate (MMTS) than Cys14 of TbTIM. The causes of this difference were explored by measuring 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 TbTIM. 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 difference could account for the different sensitivity of the enzymes to thiol reagents, we determined the k(2) of inactivation at equal levels of ionization of their interface cysteines. Under these conditions, the difference in k(2) between TcTIM and TbTIM became 8-fold, whereas that of the triple mutant to TbTIM was 1.5 times. The substrate analogue phosphoglycolate did not modify 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, under conditions in which the interface cysteines of the enzymes had equal levels of protonation, the difference in k(2) of TcTIM and TbTIM became smaller, whereas k(2) of the triple mutant was almost equal to that of TbTIM. Thus, from measurements of the reactivity of the interface cysteine in various conditions, it was possible to obtain information on the factors that control the dynamics of a portion of the dimer interface.

The effect of monovalent cations on the P: ratios and respiratory rates of rat liver mitochondria with a K+ content of 15 mM or less has been studied. The respiratory rate and the P: O ratios of these K+-depleted mitochondria oxidiz-

Some of the characteristics of unisite hydrolysis of [gamma 32P]ATP as well as the changes that occur on the transition to multisite catalysis were further studied. It was found that a fraction of [gamma 32P]ATP bound at the catalytic sites of F1 under unisite conditions undergoes both hydrolysis and release induced by medium nucleotides upon addition of millimolar concentrations of ADP or ATP. The fraction of [gamma 32P]ATP that undergoes release is similar to the fraction that undergoes hydrolytic cleavage, indicating that the rates of the release and hydrolytic reactions of bound [gamma 32P]ATP are in the same range. As part of studies on the mechanisms through which trifluoperazine inhibits ATP hydrolysis, its effect on unisite hydrolysis of [gamma 32P]ATP was also studied. Trifluoperazine diminishes the rate of unisite hydrolysis by 30-40%. The inhibition is accompanied by a nearly tenfold increase in the ratio of [gamma 32P]ATP/32Pi bound at the catalytic site and a 50% diminution in the rate of 32Pi release from the enzyme into the media. Trifluoperazine also induces heterogeneity of the three catalytic sites of F1 in the sense that in a fraction of F1 molecules, the high-affinity catalytic site has a turnover rate lower than the other two. Trifluoperazine does not modify the release of previously bound [gamma 32P]ATP induced by medium nucleotides. The latter indicates that hindrances in the release of Pi do not necesarily accompany alterations in the release of ATP even though both species lie in the same site.

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