Aconitase: its source of catalytic protons.
An ordinary isotope partition experiment was performed to determine the rate of dissociation of the proton from the donor site for the hydration of cis-aconitate. Aconitase in [3H]water was efficiently diluted into well-mixed solutions of cis-aconitate. Citrate and isocitrate that were formed within 2 s were more heavily labeled than could be explained by consideration of an isotope effect in the processing of one proton per enzyme equivalent. Control experiments indicate that mixing was much more rapid than catalytic turnover, ruling out incompletely diluted [3H]water as a significant isotope source. Therefore, it appears that significantly more than one enzyme-bound tritium atom (protons) must have been used in the course of the multiple turnover of the enzyme after the dilution was complete. Isotope incorporation reached values in excess of four proton equivalents as a limit with simple Michaelis dependence on cis-aconitate. From the half-saturation concentration value for trapping, 0.15 mM, the t 1/2 for exchange of each of these protons with solvent appears to be approximately 0.1 s at 0 degrees C. The large number of protons trapped seems to suggest the existence of a structurally stabilized pool of protons, or water, that communicates between the active site base and the medium in the hydration of cis-aconitate. The proton abstracted in the dehydration of [3H]citrate is transferred directly to undissociated cis-aconitate to form isocitrate without dilution, or cis-aconitate having dissociated, the tritium passes to the medium, presumably through the pool of bound protons indicated above. All of the citrate-derived protons can be found in isocitrate if cis-aconitate is added in sufficient concentration.(ABSTRACT TRUNCATED AT 250 WORDS)
Kuo
D J
DJ
Institute for Cancer Research, Fox Chase Cancer Center, Philadelphia, Pennsylvania 19111.
Rose
I A
IA
D000154:Aconitate Hydratase / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D007700:Kinetics; D008929:Mitochondria, Heart / Q000201:enzymology; D011485:Protein Binding; D011522:Protons; D051381:Rats; D014316:Tritium
D011522:Protons; D014316:Tritium; D000154:Aconitate Hydratase