Protein Science, Vol 5, Issue 2 320-330, Copyright © 1996 by Cold Spring Harbor Laboratory Press

ARTICLE

Reconstitution of active octameric mitochondrial creatine kinase from two genetically engineered fragments

M. GROSS, M. WYSS, E. M. FURTER-GRAVES, T. WALLIMANN and R. FURTER
Swiss Federal Institute of Technology, Institute for Cell Biology, ETH-Honggerberg, CH-8093 Zurich

Creatine kinase (CK) has been postulated to consist of two flexibly hinged domains. A previously demonstrated protease-sensitive site in M-CK (Morris & Jackson, 1991) has directed our attempts to dissect mitochondrial CK (Mi-CK) into two protein fragments encompassing amino acids [1-167] and [168-380]. When expressed separately in Escherichia coli, the two fragments yielded large amounts of insoluble inclusion bodies, from which the respective polypeptides could be purified by a simple two-step procedure. In contrast, co-expression of the two fragments yielded a soluble, active, and correctly oligomerizing enzyme. This discontinuous CK showed nearly full specific activity and was virtually indistinguishable from native Mi-CK by far- and near-UV CD. However, the positive cooperativity of substrate binding was abolished, suggesting a role of the covalent domain linkage in the crosstalk between the substrate binding sites for ATP and creatine. The isolated C-terminal fragment refolded into a native-like conformation in vitro, whereas the N-terminal fragment was largely unfolded. Prefolded [168-380] interacted in vitro with [1-167] to form an active enzyme. Kinetic analysis indicated that the fragments associate rapidly and with high affinity (1/K(1) = 17 {mu}M) and then isomerize slowly to an active enzyme (k(2) = 0.12 min(-1); k(-2) = 0.03 min(-1)). Our data suggest that the C-terminal fragment of Mi-CK represents an autonomous folding unit, and that the folding of the C-terminal part might precede the conformational stabilization of the N-terminal moiety in vivo.
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