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 M M (CK-MM, muscle-type) and BE (CK-BB, brain-type), both homodimers, an d isozyme MB (CK-MB), a heterodimer, were compared to determine how fo rmation of the hybrid modifies subunit conformation and dynamics. Circ ular dichroic spectra revealed additional a-helical content for the hy brid isozyme. Double-beam absorption difference spectra between CK-MB and a stoichiometric mixture of CK-MM and CE(-BB revealed decreased ex posure 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 aniso tropy spectra and decay of the anisotropy of CK derivatized on a singl e subunit with the fluorescent sulfhydryl reagent odoacetyl)amino-ethy l]aminonaphthalene-l-sulfonate indicated that the derivatized sites ar e more flexible in the heterodimer. The slow component in the anisotro py decay suggests that hybridization results in a small increase in th e packing density or contraction of overall conformation of the B-subu nit. The K-M for MgATP with singly derivatized CKMB was the same as th e KM for the native enzyme. However, derivatization of a single subuni t caused the V-max to decrease by greater than 50%, which indicates th at subunit-subunit interactions may modulate the activity of CK. A mod el for assembly of CK-MB is proposed which includes subunit characteri stics 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. (C) 1998 Elsevier Science B.V. All rights re served.