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 (Mib-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 > or = 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;Mib-CK.creatine.MgADP.NO3-), and Trp-223 is probably required for the conformational change leading to the TSAC-induced octamer dissociation of Mib-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 Mib-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 Mib-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.
Gross
M
M
Swiss Federal Institute of Technology, ETH-Hönggerberg, Zürich.
Furter-Graves
E M
EM
Wallimann
T
T
Eppenberger
H M
HM
Furter
R
R
D000818:Animals; D001081:Apyrase / Q000494:pharmacology; D001665:Binding Sites; D002645:Chickens; D002942:Circular Dichroism; D003402:Creatine Kinase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004492:Edetic Acid / Q000494:pharmacology; D004795:Enzyme Stability; D004926:Escherichia coli; D046911:Macromolecular Substances; D008928:Mitochondria / Q000201:enzymology*; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D012518:Sarcomeres / Q000201:enzymology; D013050:Spectrometry, Fluorescence; D014364:Tryptophan
D046911:Macromolecular Substances; D014364:Tryptophan; D004492:Edetic Acid; D003402:Creatine Kinase; D001081:Apyrase