Reaction of Transketolase-Bound DHEThDP Intermediate with Acceptor Substrate Ribose 5-phosphate

The reaction of enzyme-bound DHEThDP intermediate with increasing concentrations of acceptor substrate R5P was monitored using UV-Vis absorbance spectroscopy and double-jump stopped-flow mixing technique and analyzed quantitatively.

Fig.19: Transient for the reaction of enzyme bound DHEThDP intermediate in EcTK with acceptor substrate R5P.

Sequential stopped-flow kinetics for reaction of 1 mg/ml EcTK (13.88 µM active sites) that were initially mixed with equimolar concentration of HPA to form enzyme-bound DHEThDP intermediate with 2.5 mM R5P in 0.3 mM ThDP, 2.5 mM CaCl2, 50 mM glycylglycine (pH 7.6) at 25 °C. Measurements were performed with a stopped-flow spectrophotometer at a pathlength of 10 mm. The transient was fitted according to a single exponential equation first order (red, A = A1 * e^-k1*t + offset) and a double exponential equation first order (blue, A = A1 * e^-k1*t+ A2 * e^-k2*t+ offset). The residuals for both fits are shown color-coded below the transient.

After initial equimolar mixing of donor analogue β-hydroxypyruvate (HPA) with EcTK the DHEThDP intermediate was generated on the enzyme. In a second mixing step the so accumulated DHEThDP was rapidly mixed with acceptor substrate R5P and the reaction was monitored by UV-Vis absorbance spectroscopy. A typical stopped-flow transient for the reaction of DHEThDP intermediate with acceptor R5P is shown in Fig.19. The distribution of data points is way better represented by a double exponential than by a single exponential equation suggesting that two processes contribute to signal depletion at 300 nm:

absorbance at 300 nm

0,005 0,010 0,015 0,020 0,025

residuals

-0,0015 0,0000 0,0015

t (s)

0,00 0,02 0,04 0,06 0,08 0,10

residuals

-0,0015 0,0000 0,0015

single exponential decay double exponential decay

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a.) The very fast, first phase could monitor R5P binding whereas the second phase might represent the reaction of R5P with DHEThDP. Since binding of such a small molecule can be assumed to occur almost diffusion-controlled it is questionable if the first process monitors R5P binding.

b.) Another interpretation of biphasic behavior would be the existence of two active sites with different catalytic competence. This could be generated by negative cooperativity within the TK dimer, a controversially discussed phenomenon for ThDP-dependent enzymes (Frank et al., 2007).

c.) It is also reasonable to assume that the first phase monitors the reaction of DHEThDP with R5P and the second phase monitors the liberation of S7P (product of the reaction). This proposal is supported by kinetic measurements of EcTK with the donor F6P indicating that donor-ThDP formation is associated with an absorbance signal between 290-300 nm.

Transients were collected for a set of R5P concentrations. The rate constants for the first and second phase were plotted against the utilized R5P concentrations (Fig. 20) and fitted according to a hyperbolic equation to derive the kinetic parameters kmax and KSapp. The depletion of the DHEThDP intermediate in presence of acceptor substrates is a very fast process for EcTK wt with a kmax = 531 ± 37 s^-1. The second phase, which presumably monitors release of S7P, has a smaller signal amplitude relative to the first phase and a k^max value of 42.5 ± 2.2 s^-1.

In order to assign both phases unambiguously a ¹H-NMR based intermediate analysis in combination with rapid quenched-flow technique has to be performed for EcTK. Given that the DHEThDP intermediate in EcTK wt depletes relatively fast (kdepl = 0.0057 s^-1, Fig. 50) and that quenched-flow experiments can´t be carried out in the required sequential mixing mode such experiments are technically demanding. EcTK active site variants with prolonged life-times of the DHEThDP intermediate, which became available recently, are promising candidates for such experiments.

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Fig. 20: Kinetic analysis for the reaction of enzyme bound DHEThDP intermediate in EcTK with acceptor substrate R5P. Reaction of 1 mg/ml EcTK wt (13.88 µM active sites) that were initially mixed with equimolar concentration of HPA to form enzyme-bound DHEThDP intermediate in 0.3 mM ThDP, 2.5 mM CaCl2, 50 mM glycylglycine (pH 7.6) at 25 °C with increasing concentrations of acceptor R5P. Kinetics were initiated by 1/1 mixing and monitored in a stopped-flow absorbance spectrometer (pathlength 10 mm at 300 nm).Top left: Selected reaction transients.Top right: Simplified kinetic scheme for the experiment. Bottom: Rate constants (k^obs) were determined according to a double exponential equation (A = A1 * e^-k1*t + A2 * e^-k2*t + offset) and then plotted against the utilized R5P concentration. Kinetic parameters (k^max and K^M (R5P)) were determined according to a hyperbolic equation.

All reaction transients presented in this chapter revealed a biphasic behavior which coud be explained with the existence of two active sites with different catalytic competence. Such non-equivalence of active sites within a functional oligomer was reported for numerous ThDP-dependent enzymes (Frank et al., 2007) and Seifert et al. could demonstrate for human pyruvate dehydrogenase that elementary catalytic steps like cofactor activation, binding and conversion of substrate proceed 2-3 orders of magnitude faster in one active site relative to the second, so called, “dormant” site (Seifert et al., 2006).

Additional crystallographic and functional evidence for the catalytic non-equivalence in pyruvate dehydrogenase was presented recently (Nemeria et al., 2010). These experiments are consistent with a previously suggested model of active site synchronization or “cross-talk” via a proton wire (Frank et al., 2007; Frank et al., 2004). Such fascinating regulation of enzyme activity that fixes two active sites in

ribose 5-phosphate (mM)

0,00 0,02 0,04 0,06 0,08 0,10

absorbance at 300 nm

60

an alternating activation state was thereafter also in discussion for transketolases. However, to our knowledge no direct kinetic or structural evidence was yet presented that such a mode of regulation does exist in any transketolase. Moreover, our recent and also previous x-ray crystallographyic experiments (Asztalos et al., 2007; Fiedler et al., 2002) as well as results from NMR-based intermediate analysis revealed the formation of covalent intermediates in both active sites of the functional dimer (Asztalos et al., 2007; Mitschke et al., 2010).

Fig. 21: Summary for the microscopic kinetic analysis of the EcTK-catalyzed conversion of F6P and R5P to form S7P and E4P. Product (S7P, 42 ± 2 s^-1) release or donor cleavage are the rate limiting step of the over-all reaction (*(Sprenger et al., 1995), **(Asztalos, 2007 b), ***(Lüdtke, 2008)).

The kinetic analysis of microscopic reaction steps of EcTK indicates that the rate-limiting step of the TK over-all reaction (Fig. 21) is the liberation of donor substrates which requires the cleavage of the C-C single bond connecting cofactor and substrate. The same conclusion based on equilibrium distribution of covalent intermediates in EcTK was made recently (Asztalos et al., 2007). However, since donor cleavage could not yet been analyzed quantitatively it´s not excluded that this reaction step, which requires C-C bond cleavage as well, is rate-limiting. Unfortunately, analogous experiments with hTK failed due to precipitation of the enzyme.

≈ 350 s^-1 ** ≈ 860 ± 230 s^-1

≈ 451 ± 37 s^-1 ThDP Activated

ThDP F6P-ThDP

DHETHDP S7P-ThDP

+ R5P - R5P + F6P

- F6P

-E4P + S7P +E4P

- S7P

≈ 42 ± 2 s^-1

F6P + R5P S7P + E4P

> 150 s^-1***

kmax≈ 60-70 s^-1 *

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