THE TRYPTOPHAN RESIDUES OF MITOCHONDRIAL CREATINE-KINASE - ROLES OF TRP-223, TRP-206, AND TRP-264 IN ACTIVE-SITE AND QUATERNARY STRUCTURE FORMATION

The 5 tryptophan residues of chicken sarcomeric mitochondrial creatine kinase (Mi(b)-CK) were individually replaced by phenylalanine or cyst eine using site-directed mutagenesis. The mutant proteins were analyze d by enzyme kinetics, fluorescence spectroscopy, circular dichroism, a nd conformational stability studies. In the present work, Trp-223 is i dentified as an active-site residue whose replacement even by phenylal anine resulted in greater than or equal to 96% inactivation of the enz yme. Trp-223 is responsible for a strong (18-21%) fluorescence quenchi ng effect occurring upon formation of a transition state-analogue comp lex (TSAC; Mi(b)-CK creatine MgADP.NO3-), and Trp-223 is probably requ ired 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. Ho wever, this dimer dissociation was counteracted by TSAC formation or t he 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 anot her strong (25%) fluorescence quenching effect, which was observed upo n the TSAC-induced octamer dissociation. Substitution of Trp-264 by cy steine drastically accelerated the TSAC-induced dissociation and desta bilized the octameric structure by one-fourth of the total free intera ction energy, probably by weakening hydrophobic contacts. The roles of the other 2 tryptophan residues, Trp-213 and Trp-268, could be less w ell assigned.