Kinetic methods

2.2.3.1. Steady-state activity assay

The steady-state enzymatic activity of hTK, EcTK wild type and related active site variants were measured spectrophotometrically by a coupled assay in which native substrates xylulose 5-phosphate (X5P) and ribose 5-phosphate (R5P) were converted into sedoheptulose 7-phosphate (S7P) and glyceraldehydes 3-7-phosphate (GAP) and GAP was further converted to G3P by employing two auxiliary enzymes triose phosphate isomerase (TIM) and

sn-glycerol-54

3-phosphate: NAD⁺ 2-oxidoreductase (G3P-DH) (Kochetov, 1982). Before measurement, the auxiliary enzymes which were stored as 4.8 M ammonia sulfate sunspension had to be dialyzed overnight against 50 mM glycylglycine, pH 7.6 to minimize the effect of high concentration of ammonia sulfate on transketolase. In this process, NADH was concomitantly oxidized to NAD⁺ and the reaction was monitored at 340 nm at certain temperatures (20 °C for hTK and 30 °C for EcTK). The assay was performed in 50 mM glycylglycine, pH 7.6 buffer with a total volume of 200 µl and was further supplemented with 0.22 mM NADH, 3.6 units of sn-glycerol-3-phosphate: NAD⁺ 2-oxidoreductase/triose-phosphate isomerase, 100 µM ThDP, 5 mM CaCl2, 0.1–2 mg/ml hTK or 0.05-0.5 mg/ml EcTK, and variable concentrations of X5P and R5P. One unit is defined as the formation of 1 µmol of G3P per min. In order to determine the Km value for X5P, the concentration of R5P was kept constant at 5 mM. The Km values for R5P was not measured due to the availability of the substrate X5P.

Fig 12: Simplified scheme of the steady-state activity assay of transketolase. Xylulose 5-phosphate (X5P) and ribose 5-phosphate (R5P) are converted by transketolase into sedoheptulose 7-phosphate (S7P) and glyceraldehyd 3-phosphate (GAP) which is linked to the formation of glycerol 3-phosphate (G3P) by two auxiliary enzymes (TIM and G3P-DH). The consumption of NADH is monitored spectrophotometrically at 340 nm.

The dependence of the initial rates on the substrate concentration was analysed according to the Michaelis-Menten equation 1 or the substrate saturation equation 2.

Equation 1: Michaelis–Menten equation. Vmax = maximal velocity at substrate saturation; KM = Michaelis constant.

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Equation 2: Substrate saturation equation. Vmax = maximal velocity at substrate saturation; KM = Michaelis constant; KI = inhibition equilibrium constant of the putative substrate inhibition.

2.2.3.2 Stopped-flow kinetic methods

Transient kinetic analysis plays an indispensable role in the understanding of working mechanism of macromolecules. For enzymes, steady-state measurements can only provide the overall catalytic efficiency and substrate specificity. In order to identify and characterize individual steps in the reaction pathway, transient or pre-steady state kinetic measurements have to be employed that is where stopped-flow and chemical-quench flow methods step in.

The stopped-flow technique only has a dead time of approx. 1.5 ms which enables the visualization of fast kinetic events such as enzymatic intermediates formation. The single mixing and sequential stopped-flow measurement were performed with a stopped-flow spectrophotometer SX.20 MV at related wavelengths. To measure the time-resolved spectra of the intermediates depletion (AP, DHEThDP) or formation (IP, DHEThDP or enolate), a diode array detector was used and spectra were collected in equidistant time intervals.

Experiments were generally performed at 4 °C for hTK and 25 °C for EcTK with on optical path length of 1 cm. Related buffers were usually used as blank at any wavelength.

2.2.3.2.1 Single mixing experiments

In order to find the optimal wavelength for individual intermediate, UV-Vis difference spectra was measured and calculated first. The wavelengths for the single mixing stopped-flow experiments were chosen as follows. For hTK, the depletion of AP, formation of IP and DHEThDP were measured at 325, 295 and 400 nm, respectively. For EcTK, the AP and IP were measured at 320 and 295 nm, respectively. A series of substrate concentrations were mixed with enzyme in a 1:1 ratio in all cases. The detailed condition regarding concentration of enzymes, cofactors, substrates and buffer systems are shown in the results part.

2.2.3.2.2 Sequential mixing experiments

In order to study whether the enzyme-bound dihydroxyethyl thiamine diphosphate intermediate (DHEThDP) derived from the non-phosphorylated substrate fructose is

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chemically active or not, a double jump (sequential) stopped-flow experiment was performed with the native acceptor substrate R5P. In the first step, hTK (8 mg/ml) was mixed with donor substrate fructose (1 M) and incubate for 500 s to populate DHEThDP at the active site. The optimal concentration of enzyme and substrate and the incubation time were determined by single mixing experiments. In the second mixing step, the so accumulated DHEThDP was mixed with a series of acceptor substrate R5P and measured at 308 nm. The detailed condition regarding concentration of enzymes, cofactors, substrates and buffer systems are shown in the results part.

2.2.3.3 Temperature jump (T-jump) relaxation technique

The temperature jump relaxation technique is a convenient and common method of studying rapid reversible reactions of biological macromolecules (Crooks, 1983). By employing a short pulse of electrical discharge at high voltage, the temperature of reaction sample could rise up to 10 °C within a few microseconds, which changes the value of the initial equilibrium constant followed by relaxation to a new equilibrium at the new temperature.

In order to study the rapid proton transfer reaction of the tautomeric and ionization states of ThDP, a temperature jump experiment was applied on hTK-wt (5 mg/ml in 50 mM glycylglycine, 5 mM CaCl2, pH 7.6). Temperature jumped from 12 °C to 20 °C and the reaction was measured by UV-Vis spectroscopy at 325 nm and 294 nm for the AP and IP form, respectively. The thereof obtained progress curves were fitted either by a single exponential equation which is used for a one-step reaction or by a double exponential equation used for a two-step reaction. Samples used for T-jump experiments could be recovered and after spinning down aggregates, protein could be reused for other purposes.