Loop movement and catalysis in creatine kinase.
Recently the crystal structure of creatine kinase from Torpedocalifornica was determined to 2.1 A. The dimeric structure revealed two different forms in the unit cell: one monomer was bound to a substrate, MgADP, and the other monomer was bound to a transition-state analogue complex composed of MgADP, nitrate and creatine. The most striking difference between the structures is the movement of two loops (comprising residues 60-70 and residues 323-333) into the active site in the transition state structure. This loop movement effectively occludes the active site from solvent, and the loops appear to be locked into place by a salt bridge formed between His66 and Asp326. His66 is of particular interest as it is located within a PGHP motif conserved in all creatine kinases but not found in other guanidino kinases. We have carried out alanine-scanning mutagenesis of each of the residues in the PGHP motif and determined that only the His66 plays a significant role in the creatine kinase reaction. Although neither residue interacts directly with the substrate, the interaction His66 and Asp326 appears to be important in providing the precise alignment of substrates necessary for phosphoryl group transfer. Finally, it is clear that neither His66 nor Asp326 are responsible for the pKs observed in the pH-rate profile for HMCK.
Wang
Pan-Fen
PF
College of Pharmacy, University of Michigan, Ann Arbor, Michigan 48109, USA.
Flynn
Allen J
AJ
McLeish
Michael J
MJ
Kenyon
George L
GL
D000244:Adenosine Diphosphate / Q000737:chemistry; D000255:Adenosine Triphosphate / Q000737:chemistry; D000409:Alanine / Q000737:chemistry; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D001224:Aspartic Acid / Q000737:chemistry; D001665:Binding Sites; D002384:Catalysis; D002942:Circular Dichroism; D003402:Creatine Kinase / Q000737:chemistry* / Q000378:metabolism; D019281:Dimerization; D014644:Genetic Variation; D006639:Histidine / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D013058:Mass Spectrometry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016296:Mutagenesis; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011485:Protein Binding; D011487:Protein Conformation; D012492:Salts / Q000494:pharmacology; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D014101:Torpedo
D012492:Salts; D001224:Aspartic Acid; D006639:Histidine; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D003402:Creatine Kinase; D000409:Alanine