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

Abstract

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/K₁, = 17 μM) and then isomerize slowly to an active enzyme (k₂ = 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.