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Spectrochemical and ligand binding studies of an active mercurinitro phenol labeled creatine kinase

, : Spectrochemical and ligand binding studies of an active mercurinitro phenol labeled creatine kinase. Biochemistry 16(17): 3838-3845

The formation of several complexes between an active creatine kinase [chicken] (muscle-type) labeled with 2-mercuri-4-nitrophenol and substrates and anions, singly or in combination, elicited different nitrophenol spectral changes. Blue shifts at different wavelength (between 405-426 nm) were observed in all but 2 spectral changes. Although the Mg2+ cofactor did not produce a change, its presence in the ternary E-MgADP complex caused a perturbation different from that generated by ADP alone. Creatine does not also induce a change, but conferred pronounced effect on binding to E-MgADP and E-MgADP-nitrate complexes. In a series of complexes that ultimately make up the transition state analog, E, E-MgADP, E-MgADP-nitrate or E-MgADP-creatine, and E-MgADP-nitrate-creatine, the spectral shifts, both in the visible and UV region, were different. These varied spectral changes and the finding that the rates of reaction of 2 equiv [equivalents] of mercurinitrophenol with the native enzyme in the same series were reduced in the order, 0-, 2-, 3- or 13- and 200-fold, respectively, are manifestations of the various conformational states of creatine kinase. Anion inhibitors-chloride, nitrate or sulfate-caused hypochromic shifts at 408, 406 or 400 nm, respectively, whereas acetate, an activator, brought about a hyperchromic shift at 415 nm. Pyrophosphate inhibitor caused a 439 nm red shift. Further reaction of the nitrophenol-labeled enzyme with mercurinitrophenol, iodoacetamide or 2 equiv of methylmethanethiosulfonate resulted in the modification of another pair of cysteine residues and the loss of activity. The loss of activity occurred concomitant with structural rearrangement that was monitored by the bound chromophore. Further evidence, including the peptide mapping experiment, indicates that the chromophoric probe (at 2 equiv) reacted with 1 crysteine/subunit different from the essential thiol. The structural change that is characteristic of each E-S complex and uniquely monitored by the chromophoric probe presumably occurs elsewhere than at the active site region.

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Accession: 006460105

PMID: 20128

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