MUTATION OF CIS-PROLINE-207 IN MITOCHONDRIAL CREATINE-KINASE TO ALANINE LEADS TO INCREASED ACID STABILITY

We show that the mutation of an uncharged residue far from the active site to another uncharged residue can have effects on the active site without disturbing the overall structure of the protein. Cis-proline 2 07 of mitochondrial creatine kinase was mutated to alanine, The mutant showed a decrease in the pH-optimum for ATP synthesis by 1.5 units wh ile the maximum relative activity was lowered to 53% of the wild-type enzyme. In the direction of ATP consumption, the pH optimum was lowere d by 1.3 units and the maximum relative activity was 49% of the wildty pe enzyme, The enzyme kinetic parameters K-m and K-d for the substrate s did not change dramatically, indicating a largely unperturbed active site. Small-angle X-ray scattering was used to investigate the struct ural change concomitant with the mutation, yielding a scattering profi le only slightly different from that of the wild-type enzyme. Neither the radius of gyration nor the molecular mass showed any significant d ifferences, leading to the conclusion that quarternary organization an d fold of the mutant and the wild-type enzymes were similar. Theoretic al analysis suggests the most probable primary source of structural ch ange to be a transition of residue 207 peptide bond torsional angle om ega from the cis to the trans configuration.