Phosphoenolpyruvate and Mg2+ binding to pyruvate kinase monitored by infrared spectroscopy

Abstract

Structural changes in rabbit muscle pyruvate kinase (PK) induced by phosphoenolpyruvate (PEP) and Mg(2+) binding were studied by attenuated total reflection Fourier transform infrared spectroscopy in combination with a dialysis accessory. The experiments indicated a largely preserved secondary structure upon PEP and Mg(2+) binding but also revealed small backbone conformational changes of PK involving all types of secondary structure. To assess the effect of the protein environment on the bound PEP, we assigned and evaluated the infrared absorption bands of bound PEP. These were identified using 2,3-(13)C(2)-labeled PEP. We obtained the following assignments: 1589 cm(-1) (antisymmetric carboxylate stretching vibration); 1415 cm(-1) (symmetric carboxylate stretching vibration); 1214 cm(-1) (C-O stretching vibration); 1124 and 1110 cm(-1) (asymmetric PO(3)(2-) stretching vibrations); and 967 cm(-1) (symmetric PO(3)(2-) stretching vibration). The corresponding band positions in solution are 1567, 1407, 1229, 1107, and 974 cm(-1). The differences for bound and free PEP indicate specific interactions between ligand and protein. Quantification of the interactions with the phosphate group indicated that the enzyme environment has little influence on the P-O bond strengths, and that the bridging P-O bond, which is broken in the catalytic reaction, is weakened by <3%. Thus, there is only little distortion toward a dissociative transition state of the phosphate transfer reaction when PEP binds to PK. Therefore, our results are in line with an associative transition state. Carboxylate absorption bands indicated a maximal shortening of the length of the shorter C-O bond by 1.3 pm. PEP bound to PK in the presence of the monovalent ion Na(+) exhibited the same band positions as in the presence of K(+), indicating very similar interaction strengths between ligand and protein in both cases.

Figure 1

Chemical structures of (a) PEP and (b) pyruvate. Asterisks indicate the ¹³C-labeled carbon atoms in panel a.

Figure 2

Infrared spectra of (a) 100 mM unlabeled PEP, (b) 50 mM labeled PEP, and (c) 100 mM pyruvate dissolved in H₂O at pH 7.5.

Figure 3

Infrared absorbance changes of PK upon PEP binding in the presence of K⁺ and Mg²⁺ in H₂O. Traces a–c reflect 96, 180, and 240 s spectra after addition of 25 μM PEP. Traces d and e show spectra of the subsequent additions of 25 μM PEP measured 240 s after each addition. The spectra have been shifted vertically for a clearer presentation.

Figure 4

Evolution of the 970 cm⁻¹ band after addition of PEP to PK. (a) Series of overlaid spectra showing the band of the symmetric PO₃^2- stretching vibration after the first PEP addition. The spectra were recorded in H₂O within 240 s after the addition of PEP and were smoothed over 25 cm⁻¹. The band amplitude increased with time and the band position shifted. (b) Time course of the band position after the first (I) and the second addition (II) of PEP. Spectra were smoothed over 25 data points before evaluation of the band position. Kinetics of the IR absorbance change of PK, monitored by integrated band intensities at 1695 cm⁻¹, upon PEP binding in H₂O (c).

Figure 5

Binding-induced absorbance changes of PK and ligand. All spectra were recorded 84–144 s after the addition of ligand and normalized to the amplitude of the amide I signals in the 240 s spectrum. (a and b) Binding of unlabeled PEP in the presence of K⁺ and Mg²⁺ in (a) H₂O and (b) D₂O. (c) Binding of [2,3-13C] PEP in H₂O. (d) Binding of PEP in the presence of Na⁺ and Mg²⁺ in H₂O. (e and f) Mg²⁺ binding to PK in the presence of K⁺ in (e) H₂O and (f) D₂O. Band positions are labeled in panels b–d if they differ from the respective positions in panel a. In panel f, only those bands are labeled that have a different position in panel e. The spectra have been shifted vertically for a clearer presentation.

Figure 6

Enzymatic reaction of PK. Changes of IR absorbance induced by addition of PEP to PK and ADP. The initial state of the sample was a mixture of PK·Mg²⁺·K⁺ and ADP, then PEP was added. The spectrum shown is averaged over the first 4 min after PEP addition.