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The tryptophan residues of mitochondrial creatine kinase: Roles of Trp‐223, Trp‐206, and Trp‐264 in active‐site and quaternary structure formation

1994; Wiley; Volume: 3; Issue: 7 Linguagem: Inglês

10.1002/pro.5560030708

1469-896X

Martin Groß, Elizabeth M. Furter‐Graves, Theo Wallimann, Hans M. Eppenberger, Rolf Furter,

Protein Structure and Dynamics

Abstract The 5 tryptophan residues of chicken sarcomeric mitochondrial creatine kinase (Mi b ‐CK) were individually replaced by phenylalanine or cysteine using site‐directed mutagenesis. The mutant proteins were analyzed by enzyme kinetics, fluorescence spectroscopy, circular dichroism, and conformational stability studies. In the present work, Trp‐223 is identified as an active‐site residue whose replacement even by phenylalanine resulted in ≥96% inactivation of the enzyme. Trp‐223 is responsible for a strong (18–21%) fluorescence quenching effect occurring upon formation of a transition state–analogue complex (TSAC; Mi b ‐CK·creatine·MgADP·NO 3 – ), and Trp‐223 is probably required for the conformational change leading to the TSAC‐induced octamer dissociation of Mi b ‐CK. Replacement of Trp‐206 by cysteine led to a destabilization of the active‐site structure, solvent exposure of Trp‐223, and to the dissociation of the Mi b ‐CK dimers into monomers. However, this dimer dissociation was counteracted by TSAC formation or the presence of ADP alone. Trp‐264 is shown to be located at the dimer–dimer interfaces within the Mi b ‐CK octamer, being the origin of another strong (25%) fluorescence quenching effect, which was observed upon the TSAC‐induced octamer dissociation. Substitution of Trp‐264 by cysteine drastically accelerated the TSAC‐induced dissociation and destabilized the octameric structure by one‐fourth of the total free interaction energy, probably by weakening hydrophobic contacts. The roles of the other 2 tryptophan residues, Trp‐213 and Trp‐268, could be less well assigned.