Structural Properties of the Creatine‐Kinase Active Site Studied by Chromophoric‐Reagent Labelling

Abstract

The environment of the active site of creatine kinase has been investigated by means of three chromophoric reagents: 2‐hydroxy‐5‐nitrobenzyl bromide, 2‐methoxy‐5‐nitrobenzyl bromide and 2‐(dansylamino)ethyl monophosphate. The first two reagents are incorporated at the essential thiol groups. The effect of pH on the spectra of the 2‐hydroxy‐5‐nitrobenzyl‐labelled creatine kinase suggests that this reagent lies inside the active site near an ionized residue, while the 2‐methoxy‐5‐nitrobenzyl group is apparently exposed to the medium. Conformational changes induced by the binding of different ligands, including nucleotide substrates or anions, produce only perturbations of the 2‐hydroxy‐5‐nitro‐benzyl chromophore. The 2‐(dansylamino)ethyl monophosphate (which is really an analogue of the nucleotide substrate) interacts with the nucleotide site of creatine and arginine kinases in a relatively hydrophobic environment.