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)