ATP Nucleotidylation of Creatine Kinase

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Abstract
Creatine kinase (CK) will autoincorporate radiolabel from [γ32P]ATP and has thus been reported to be autophosphorylated. Also, in contrast to normal brain enzyme, CK in Alzheimer-diseased brain homogenate shows greatly decreased activity, abolished photolabeling with [32P]8N3ATP, and no detectable autoincorporation of radiolabel by [γ32P]ATP. Surprisingly, our studies with both human brain and purified CK showed that [α32P]ATP, [γ32P]ATP, [α32P]ADP, [2,8H3]ATP, [γ32P]2‘,3‘-O-(2,4,6-trinitrophenyl)-ATP, and [γ32P]benzophenone-γATP all autoincorporate radiolabel into CK with good efficiency. This demonstrates that the γ-phosphate and the 2‘ and 3‘ hydroxyls are not involved in the covalent linkage and that all three phosphates, the ribose and base of the ATP molecule are retained upon autoincorporation (nucleotidylation). Treatment with NaIO3 to break the 2‘−3‘ linkage effected total loss of radiolabel indicating that nucleotidylation resulted in opening of the ribose ring at the C1‘ position. Nucleotidylation with increasing [α32P]ATP at 37 °C gives an approximate k0.5 of 125 μM and saturates at 340 μM nucleotide. Modification of 8−10% of the copy numbers occurs at saturation, and CK activity is inhibited to approximately the same degree. Low micromolar levels of native substrates such as ADP, ATP, and phosphocreatine substantially reduce [α32P]ATP nucleotidylation. In contrast, AMP, GTP, GMP, NADH, and creatine did not effectively reduce nucleotidylation. When [α32P]ATP-nucleotidylated or [α32P]8N3ATP-photolabeled CK is treated with trypsin a single, identical radiolabeled peptide (V279-R291) is generated that comigrates on reverse phase HPLC and Tris-tricine electrophoresis. Nucleotidylation into this peptide was prevented 86% by the presence of ATP. We conclude that CK is nucleotidylated within the active site by modification at the C1‘position and that autophosphorylation of this enzyme does not occur.