The Structural Isomerisation of Human‐Muscle Adenylate Kinase as Studied by 1H‐Nuclear Magnetic Resonance

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Abstract
Human muscle adenylate kinase was studied by 1H-NMR spectroscopy. The C-2 and C-4 proton resonances of the active-center histidine His-36 could be identified; the pK of His-36 was determined as 6.1. The pK of His-189 is very low (4.9) although it is located at the surface of the protein. Other resonance lines are discussed in comparison with NMR spectra of porcine adenylate kinase. A pH-dependent structural isomerization as shown by X-ray crystallography in the pig enzyme was not observed for human adenylate kinase in solution. However, the binding of adenosine(5'')pentaphospho(5'')adenosine (Ap5A), a bisubstrate inhibitor, to adenylate kinase causes an overall change of the NMR spectrum indicative of a large conformational change of the enzyme. The exchange rate (koff) for Ap5A was estimated as 10 s-1 and decreases by addition of Mg2+. On the basis of these values and the known Kd it is likely that the binding of Ap5A is a diffusion-controlled process, kon being 108 M-1 s-1. In conclusion, the system Ap5A/Mg2+/human adenylate kinase, which was studied by NMR spectroscopy and X-ray diffraction in parallel, is suitable for analyzing the induced fit postulated by Jencks for all kinase-catalyzed reactions.