Abstract
Abstract The kinetic behavior of rhodanese (thiosulfate:cyanide sulfurtransferase, EC 2.8.1.1) was investigated at pH values from 5.0 to 10.8. The binding of thiosulfate was shown to be dependent upon a pK' of 9.9, with the protonated enzyme binding the substrate much more strongly than the deprotonated form. An enzymic nucleophile displaces sulfite to form the sulfur-substituted enzyme. This reaction was shown to be dependent upon a pK' of 6.5, with the protonated form being unreactive. The sulfur-substituted enzyme has three forms which are related by pK' values of 5.9 and 9.4. The second order rate constants for the reaction of these enzymic forms with cyanide ion are 8.9 x 108 m-1 s-1, 2 x 107 m-1, s-1, and ≤104 m-1 s-1, as one goes from the most protonated to the least protonated form. The activation parameters for this reaction were studied at pH 8.7. The activation enthalpy was very small while the activation entropy was large and negative, probably indicating that there is only a transition state between the sulfur-substituted enzyme and the products and that the transition state involves considerable charge neutralization. The free enzyme and the sulfur-substituted enzyme are known to bind numerous anionic species, including substrates, which inhibit the reaction. The pH dependence of this inhibition indicated the importance of the same enzymic forms as the studies of the catalyzed reaction itself. This formal mechanism, combined with previous results, suggests a chemical interpretation on the following basis. (a) The enzyme contains a divalent cationic site which has at least one catalytically active water ligand. (b) Thiosulfate binding displaces one water ligand; the loss of a proton from this water ligand prevents both thiosulfate binding and the reaction with cyanide anion. (c) The enzymic nucleophile which displaces sulfite is a cysteine mercaptolate anion that is inactivated by protonation; this sulfhydryl may be either a direct ligand to the cationic site or hydrogen bonded to it, possibly through a second water ligand. (d) The sulfur-substituted enzyme has the cysteine persulfide structure as a ligand with the cationic site and this rapidly reacts with an outer sphere complex that the cationic site forms with cyanide. (e) Inhibitory anions displace a water ligand at the cationic site and decrease the enzyme-substrate interaction by mechanisms similar to those of the high pH ionizations of the free enzyme and the sulfur-substituted enzyme. The proposed chemical mechanism is consistent both with the results presented here and with the previous information concerning the enzyme.
Highlights
The slopes or intercepts of such primary plots were used to construct cyanide or thiosulfate secondary plots. In this manner the rate and equilibrium constants evaluated in this study were obtained
PH Dependence of Rate and Equilibrium Constants-Inspection of Equation 1 shows that the pH dependence of the thiosulfate slope depends upon the pH dependencies of K, and k+z
The experimental results show that the value of k+t depends upon a single ionization, pKESsoJ = 6.5, which was determined graphically (Fig. 3)
Summary
The chemicals used as buffer components and substrates were all reagent grade materials from J. T. Baker Chemical Co. and were not further purified. Rhodanese was prepared from frozen bovine liver as described by Horowitz and DeToma [15]. In all preparations the crystalline suspension of rhodanese had an activity and a protein concentration consistent with purit,y greater than 95%. These crystals were dissolved to make a stock enzyme solution, the titer of which was determined as described by Wang and Volini [12]. Stock solutions were stable for at least a week with less than 10% loss of activity if they were frozen when not being used
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