Use of creatine kinase to induce multistep reactions in infrared spectroscopic experiments.
An extension of current approaches to trigger enzymatic reactions in reaction-induced infrared difference spectroscopy experiments is described. A common procedure is to add a compound that induces a reaction in the protein of interest. To be able to induce multistep reactions, we explored here the use of creatine kinase (CK) for the study of phosphate transfer mechanisms. The enzymatic reaction of CK could be followed using bands at 1614 and 979 cm(-1) for creatine phosphate consumption, at 944 cm(-1) for ADP consumption, and at 1243, 992, and 917 cm(-1) for ATP formation. The potential of CK to induce multistep reactions in infrared spectroscopic experiments was demonstrated using the sarcoplasmic reticulum Ca(2+)-ATPase (SERCA1a) as the protein of interest. ADP binding to the ATPase was triggered by photolytic release of ADP from P(3)-1-(2-nitro)phenylethyl ADP (caged ADP). CK added in small amounts converted the released ADP to ATP on the time scale of minutes. This phosphorylated the ATPase and led to the formation of the first phosphoenzyme intermediate Ca2E1P. Thus a difference spectrum could be obtained that reflected the reaction from the ADP ATPase complex to the first phosphoenzyme intermediate. Comparison with a phosphorylation spectrum obtained when the initial state was the ATP ATPase complex revealed the contribution of ATP's γ-phosphate to the conformational change of the ATPase upon nucleotide binding: γ-phosphate binding modifies the structure of a β-sheet, likely in the phosphorylation domain, and shifts its spectral position from ~1640 to ~1630 cm(-1). Upon phosphorylation of the ATPase, the β-sheet relaxes back to a structure that is intermediate between that adopted in the ADP bound state and that in the ATP bound state.
Department of Biochemistry and Biophysics, The Arrhenius Laboratories for Natural Sciences, Stockholm University , SE-106 91 Stockholm, Sweden.
Nadejda
N
Eremina
Andreas
A
Barth
D000818:Animals; D055162:Biocatalysis; D003402:Creatine Kinase / Q000737:chemistry / Q000378:metabolism*; D004789:Enzyme Activation; D007700:Kinetics; D009132:Muscles / Q000201:enzymology; D011817:Rabbits; D013055:Spectrophotometry, Infrared
D003402:Creatine Kinase