THE ACTIVE-SITE HISTIDINES OF CREATINE-KINASE - A CRITICAL ROLE OF HIS-61 SITUATED ON A FLEXIBLE LOOP

A histidine residue with a pK(a) of 7 has been inferred to act as a ge neral acid-base catalyst for the reaction of creatine kinase (CK), cat alyzing the reversible phosphorylation of creatine by ATP. The chicken sarcomeric muscle mitochondrial isoenzyme Mi(b)-CK contains several h istidine residues that are conserved throughout the family of creatine kinases. By X-ray crystal structure analysis, three of them (His 61, His 92, and His 186) were recently shown to be located close to the ac tive site of the enzyme. These residues were exchanged against alanine or aspartate by in vitro mutagenesis, and the six mutant proteins wer e expressed in E. coli and purified. Structural integrity of the mutan t proteins was checked by small-angle X-ray scattering. Kinetic analys is showed the mutant His 61 Asp to be completely inactive in the direc tion of ATP consumption while exhibiting a residual activity of 1.7% o f the wild-type (wt) activity in the reverse direction. The respective His to Ala mutant of residue 61 showed approximately 1% wt activity i n the forward and 10% wt activity in the reverse reaction. All other m utants showed near wt activities. Changes in the kinetic parameters K- m or V-max, as well as a significant loss of synergism in substrate bi nding, could be observed with all active mutants. These effects were m ost pronounced for the binding of creatine and phosphocreatine, wherea s ATP or ADP binding were less severely affected. Based on our results , we assume that His 92 and His 186 are involved in the binding of cre atine and ATP in the active site, whereas His 61 is of importance for the catalytic reaction but does not serve as an acid-base catalyst in the transphosphorylation of creatine and ATP. In addition, our data su pport the idea that the flexible loop bearing His 61 is able to move t owards the active site and to participate in catalysis.