Phosphoenolpyruvate and Mg2+ binding to pyruvate kinase monitored by infrared spectroscopy.
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.
Kumar
Saroj
S
Department of Biochemistry and Biophysics, Stockholm University, Stockholm, Sweden.
Barth
Andreas
A
D000042:Absorption; D000818:Animals; D055162:Biocatalysis; D004789:Enzyme Activation / Q000187:drug effects; D008274:Magnesium / Q000378:metabolism* / Q000494:pharmacology; D010728:Phosphoenolpyruvate / Q000378:metabolism* / Q000494:pharmacology; D011485:Protein Binding; D011487:Protein Conformation / Q000187:drug effects; D011770:Pyruvate Kinase / Q000737:chemistry / Q000378:metabolism*; D011817:Rabbits; D012964:Sodium / Q000494:pharmacology; D013055:Spectrophotometry, Infrared
D010728:Phosphoenolpyruvate; D012964:Sodium; D011770:Pyruvate Kinase; D008274:Magnesium