Subunit conformation and dynamics in a heterodimeric protein: studies of the hybrid isozyme of creatine kinase.
Several physical properties of creatine kinase (EC 2.7.3.2) isozymes MM (CK-MM, muscle-type) and BB (CK-BB, brain-type), both homodimers, and isozyme MB (CK-MB), a heterodimer, were compared to determine how formation of the hybrid modifies subunit conformation and dynamics. Circular dichroic spectra revealed additional alpha-helical content for the hybrid isozyme. Double-beam absorption difference spectra between CK-MB and a stoichiometric mixture of CK-MM and CK-BB revealed decreased exposure of intrinsic chromophores in the hybrid. The relative intensity of the intrinsic fluorescence of CK-MB was between the two homodimers, but was 16% closer to the less fluorescent CK-MM. Steady state anisotropy spectra and decay of the anisotropy of CK derivatized on a single subunit with the fluorescent sulfhydryl reagent 5-[2-(iodoacetyl)amino-ethyl]aminonaphthalene-1-sulfonate indicated that the derivatized sites are more flexible in the heterodimer. The slow component in the anisotropy decay suggests that hybridization results in a small increase in the packing density or contraction of overall conformation of the B-subunit. The KM for MgATP with singly derivatized CK-MB was the same as the KM for the native enzyme. However, derivatization of a single subunit caused the Vmax to decrease by greater than 50%, which indicates that subunit-subunit interactions may modulate the activity of CK. A model for assembly of CK-MB is proposed which includes subunit characteristics more similar to those found in the muscle-type homodimer than in the brain-type homodimer and increased flexibility of the active site domain of both subunits.
Grossman
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SH
Department of Chemistry, University of South Florida, Tampa, FL 33620, USA. stgr@chuma.cas.usf.edu
Sellers
D S
DS
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D001921:Brain / Q000201:enzymology; D002942:Circular Dichroism; D003402:Creatine Kinase / Q000737:chemistry*; D019281:Dimerization; D005454:Fluorescence Polarization; D005456:Fluorescent Dyes / Q000378:metabolism; D007527:Isoenzymes / Q000737:chemistry; D007700:Kinetics; D009132:Muscles / Q000201:enzymology; D009206:Myocardium / Q000201:enzymology; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011817:Rabbits; D013053:Spectrophotometry
D005456:Fluorescent Dyes; D007527:Isoenzymes; D000255:Adenosine Triphosphate; D003402:Creatine Kinase