Aconitase, source of catalytic protons

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Abstract
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-actonitate. 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 t1/2 for exchange of each of these protons the half-saturation concentration value for trapping, 0.15 mM, the t1/2 for exchange of each of these protons with solvent appears to be .apprx. 0.1 s at 0.degree. 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. Therefore, the abstracted proton dissociates slowly, if at all, from the enzyme in all intermediates except those from which cis-aconitate has dissociated. Half of the citrate-derived proton is trapped by .apprx. 1 mM cis-aconitate with a Michaelian dependence. Citrate, as well as trans-aconitate and tricarballylate, competes with cis-aconitate in its utilization of the citrate-derived proton. Unlike cis-aconitate, these acids form complexes from which the citrate-derived proton can dissociate, and in the case of citrate, a functional complex results. The rate and mechanism of proton dissociation from E-H+ .cntdot. citrate are unknown.