2. Materials and Methods
3.1. Observation and characterization of a low barrier hydrogen bond (LBHB) in hTK
3.1.7. Pre-steady-state kinetic analysis by stopped-flow spectroscopy
In order to analyse the function of the LBHB for the donor half reaction of transketolase catalysis, single-mixing stopped-flow spectroscopy was employed to monitor the depletion of
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AP at 325 nm and the formation of IP at 295 nm when human TK is rapidly mixed with the donor substrate F6P. The reaction transients measured at 325 nm for the E160Q and E160A variants revealed a biphasic process while a single phase was observed for the wild type (Fig 24). The second phase ambiguously represents the depletion of the AP form when substrate-ThDP intermediate is accumulated on the enzyme, but the chemical origin of the first phase is not very clear yet. Plotting of the kobs of the first phase at 325 nm against the utilized F6P concentration revealed a hyperbolic dependence for both E160Q and E160A variants.
Maximal kobs of 103.61 ± 5.66 s-, Ksapp of 2.94 ± 0.40 mM and maximal kobs of 100.13 ± 4.81 s- and Ksapp of 0.39 ± 0.09 mM were obtained for the two variants respectively (Fig 25). Kinetic parameters of the fast pre-equilibrium phase for the IP form measured at 295 nm could not been obtained due to the scattered data points.
Fig 24: Representative progress curves of the pre-steady-state kinetic analysis of hTK and LBHB-related active site variants. Reaction conditions refer to Fig 26. Saturation donor substrate F6P (10 mM) were used for these reaction transients. a.) Normal scale representation for the reaction progress curves of the donor half reaction of hTK wild type, E160Q and E160A mixed with substrate F6P (10 mM) measured at 325 nm at 4 oC. Please note that the two variants undergo a two-phase reaction while only one phase was observed on the wild type. b.) Logarithmic scale representation for the same reaction transients.
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Fig 25: The first phase of the pre-steady-state kinetics for human TK E160Q and E160A. The rate constants of the first fast phase of the donor half reaction for hTK E160Q (left) and E160A (right) measured at 325 nm are plotted against the utilized F6P concentrations. Hyperbolic dependency is observed and the kobsmax and Ksapp are indicated for both variants.
Equation 3: hyperbolic equation used for the analysis of the donor half reaction of hTK with substrate F6P. Ksapp = the dissociation constant of the pre-equilibrium. k-2 = the rate constant of the carbonyl elimination. kobsmax = first order rate constant at saturation substrate concentration.
Equation 4: Hill equation used for the analysis of the donor half reaction of hTK with substrate F6P. Ksapp = the dissociation constant of the pre-equilibrium. k-2 = the rate constant of the carbonyl elimination. kobsmax = first order rate constant at saturation substrate concentration. n = Hill coefficient, when n < 1, there is negative cooperativity, when n = 1, there is no cooperativity, when n > 1, there is positive cooperativity.
Plotting of the rate constant for the second phase against the utilized F6P concentration are shown in Fig 26. The data points are better fitted according to the Hill equation (equation 4) than the normal hyperbolic equation (equation 3). The LBHB containing wild type exhibits a 2-fold velocity increase relative to the E160Q variant in which the LBHB is replaced by an ordinary hydrogen bond. A decrease of substrate affinity is also observed on the E160Q mutant (wild type, 0.66 ± 0.06 mM; E160Q, 0.94 ± 0.07 mM). The dissociation of the covalent F6P-ThDP intermediate (k-2) has drops 5-fold in the variant as well (Table 5). The most important observation lies on the cooperativity for the donor substrate F6P binding. The Hill coefficient
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of wild type (1.56 ± 0.26 for AP depletion, 1.49 ± 0.43 for IP formation) strongly indicates positive cooperativity while no cooperativity could be detected for the E160Q variant. The reaction constant for the E160A mutant lies between wild type and E160Q and the substrate affinity is similar to the wild type, suggesting that the binding for the donor half reaction is not affected by the abolishment of residue Glu160. Although structural data is not available for the E160A variant, we could deduce that a water molecule probably replaces the glutamate at the same position and acts as an acid-base catalyst. The crystal structure of the same variant of E.coli TK has been determined and a water molecule is observed at this position (data not published).
Fig 26: Pre-steady-state kinetic analysis of hTK. The donor half reaction (also named pre-steady-state) of hTK wild type and LBHB-related active site variants were measured by single-mixing stopped-flow spectroscopy. Typical reactions were performed by mixing 4 mg/ml hTK with increasing concentration of fructose 6-phosphate (F6P) in 5 mM CaCl2, 50 mM glycylglycine (pH 7.6) and measured by UV-Vis spectrometer at specific wavelength at 4 °C. Reaction constants were determined according to a single exponential equation (A = A1*e-k*t + offset). a.) Rate constants for the depletion of AP form measured at 325 nm were plotted against the utilized F6P concentration and the data points were analysed according to a Hill equation. b.) Rate constants for the formation of IP form measured at 295 nm were plotted against the utilized F6P concentration and the data points were analysed according to a Hill equation.
The formation the IP form measured at 295 nm revealed identical kinetic features thus reflects a same chemical process that happened at both wavelengths. Again, a biphasic reaction transient was observed for all three measured proteins. The values of the maximal kobs for all three proteins are slightly higher than that of the depletion of AP form, which could be partially explained by the knowledge that aromatic amino acids such as Tyr, Trp and Phe also have absorbance at this wavelength.
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Table 5: Overview of the pre-steady-state kinetic constants for hTK wild type and LBHB-related active site variants. The constants for wild type, E160Q and E160A were obtained by fitting the data points according to the Hill equation as explained before. k-2 = rate constant of the reverse reaction for the formation of F6P-ThDP covalent intermediate; kobsmax = first order rate constant at saturation substrate concentration; Ksapp = apparent equilibrium constant for the formation of F6P-ThDP covalent intermediate; The standard deviation from the mean values of triplicates are given.
Protein k-2 (s-1) kobsmax