3.2.2 Analysis of the BaSucP reaction
As was the case with Lm SucP, the base mechanism M1 provided an excellent description of the reaction of Ba SucP with sucrose and glycerol (Figure S2, panels A1 – E1). The dependence of\(\frac{v_{X}}{v_{H}}\) on [GOH] showed a 1.7-fold larger slope (=TC) for Ba SucP compared to Lm SucP. TheTC determined directly from reactions of Ba SucP at high and low [G1P] was 6.3 (± 0.1) M-1 and 8.1 (± 0.1) M-1, respectively. Evidence that theTC was dependent on both the type and the concentration of donor substrate effectively ruled out the applicability of mechanisms M1 and M2.
Fit with mechanism M3 generally yielded useful representations of all subsets of the data (Figure S2, panels A2 – E1 and C3 – E3). Peculiarities already noted for reactions of Lm SucP, like the dependence of \(v_{X}\) on [GOH] developing curvature at low [G1P] (Figure S2, panel C3 compared to panel C2), were reproduced very well by the model. Despite some disagreement between model and experiment in absolute numbers (hence the relatively small \(R^{2}\) of 0.804) (Figure 4A), mechanism M3 nonetheless captured fully the pronounced deviation from Michaelis-Menten behavior in the dependence of\(v_{H}\) on [G1P] when no glycerol was present. The dependence is biphasic. The initial “high affinity” phase at low substrate concentration (≤ 4 mM) exhibits a steep increase in \(v_{H}\) with [G1P]. However, rather than reaching plateau at high substrate concentration, the dependence changes into a second “low affinity” phase in which \(v_{H}\) again increases with increasing [G1P], yet at a much shallower slope. The biphasic dependence of \(v_{H}\) on [G1P] accords very well with mechanism M3, given that binding constants for high- and low-affinity substrate binding differ by a large amount. Indeed, we estimate from the fits that \(K_{G1PH2}\) exceeds\(K_{G1PH}\) (= 2.4 mM) by many orders of magnitude. The apparent “activation” of Ba SucP at high [G1P] (Figure 4A) marks an important distinction between this enzyme and Lm SucP which is inhibited by high [G1P] (Figure 3, panel A2). Large difference in the value of \(k_{+7^{\prime}}\) (Lm SucP: 3.0 s-1;Ba SucP: ≥ 103 s-1) is responsible for it. The full set of microscopic rate constants obtained from almost unconstrained fit (only restriction: 0.0 <\(k_{-3}\) < 0.0005 s-1) of mechanism M1 and M3 to the data and the corresponding kinetic parameters are summarized in the Supporting Information Tables S7 and S8.
Analogously as with Lm SucP, mechanism M3 predicts that, when G1P is used as the donor in reactions of Ba SucP, the TCwill increase with decreasing donor concentration to a limiting value identical with the TC of the reaction with sucrose. The experimental data show this TC change exactly (Figure 4B). Other features in which Ba SucP is noteworthy different fromLm SucP are that hydrolysis of E-Glc is slower by 2.6- to 3.0-fold; in the absence of glycerol, binding to sucrose is 4-fold tighter whereas binding to G1P is 2.6-fold weaker; and the TCis ~2-fold higher at all conditions used (donor type and concentration).