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 tau1, tau3, and tau4) were detected during multiple-guanosine turnover stopped-flow absorbance spectroscopy, in addition to a transient (tau2) 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 tau1 and tau2 are assigned to chemistry based on rapid-quench results. For tau1, the rate of chemistry is ca. 3000-fold faster than kcat (1-2 orders of magnitude greater than previous estimates). The pH dependencies of the burst amplitudes for tau1 and tau2 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 tau1 and tau2 are surprisingly pH-independent, given the prominent role of acid-base chemistry in the proposed mechanism for TvNH. tau3 and tau4 are assigned to the substrate binding and base release processes, respectively, and shown to be equivalent to two fluorescence transients (tau3 and tau4, 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.
Vandemeulebroucke
An
A
Department of Ultrastructure, Vlaams Interuniversitair Instituut voor Biotechnologie, Vrije Universiteit Brussel, Pleinlaan 2, 1050 Brussel, Belgium.
Versées
Wim
W
Steyaert
Jan
J
Barlow
John N
JN
D000818:Animals; D001665:Binding Sites; D006147:Guanine / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D007288:Inosine / Q000737:chemistry; D007700:Kinetics; D008956:Models, Chemical; D009699:N-Glycosyl Hydrolases / Q000737:chemistry* / Q000378:metabolism*; D015800:Protozoan Proteins / Q000737:chemistry* / Q000378:metabolism*; D013053:Spectrophotometry; D013379:Substrate Specificity; D016689:Trypanosoma vivax / Q000201:enzymology
D015800:Protozoan Proteins; D007288:Inosine; D006147:Guanine; D009699:N-Glycosyl Hydrolases