Multiple transients in the pre-steady-state of nucleoside hydrolase reveal complex substrate binding, product base release, and two apparent rates of chemistry

We have investigated the transient kinetics of the nucleoside hydrolase from Trypanosoma ViVax (TVNH) at low temperatures (5 degrees C). Three novel absorbance transients (termed tau 1, tau 3, and tau 4) were detected during multiple-guanosine turnover stopped-flow absorbance spectroscopy, in addition to a transient (tau 2) that had been observed previously at 35 degrees C. At 5 degrees C, TvNH displays full-sites activity and not half-of-the-sites activity as is apparent at 35 degrees C. Both tau 1 and tau 2 are assigned to chemistry based on rapid-quench results. For tau 1, the rate of chemistry is ca. 3000-fold faster than k(cat) (1-2 orders of magnitude greater than previous estimates). The pH dependencies of the burst amplitudes for tau 1 and tau 2 indicate that these transients arise from the formation of two different dimeric TVNH, substrate complexes and not from TVNH that contains kinetically asymmetric monomers. The saturating burst rates for tau 1 and tau 2 are surprisingly pH-independent, given the prominent role of acid-base chemistry in the proposed mechanism for TVNH. tau 3 and tau 4 are assigned to the substrate binding and base release processes, respectively, and shown to be equivalent to two fluorescence transients (tau 3* and tau 4*, respectively) observed previously by stopped-flow methods at 35 degrees C. The rate of base release is shown to be an apparent rate. Together with steady-state product inhibition results, the data indicate that TVNH follows an ordered uni-bi kinetic mechanism with a TVNH.base dead- end complex, and not the rapid equilibrium random uni-bi mechanism proposed for other NHs. Two applicable kinetic models are presented and their implications for future mechanistic studies discussed.