Insights into the Phosphoryl Transfer Mechanism of Human Ubiquitous Mitochondrial Creatine Kinase

Sci Rep. 2016 Dec 2:6:38088. doi: 10.1038/srep38088.

Abstract

Human ubiquitous mitochondrial creatine kinase (uMtCK) is responsible for the regulation of cellular energy metabolism. To investigate the phosphoryl-transfer mechanism catalyzed by human uMtCK, in this work, molecular dynamic simulations of uMtCK∙ATP-Mg2+∙creatine complex and quantum mechanism calculations were performed to make clear the puzzle. The theoretical studies hereof revealed that human uMtCK utilizes a two-step dissociative mechanism, in which the E227 residue of uMtCK acts as the catalytic base to accept the creatine guanidinium proton. This catalytic role of E227 was further confirmed by our assay on the phosphatase activity. Moreover, the roles of active site residues in phosphoryl transfer reaction were also identified by site directed mutagenesis. This study reveals the structural basis of biochemical activity of uMtCK and gets insights into its phosphoryl transfer mechanism.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adenosine Triphosphate / metabolism
  • Amino Acid Substitution
  • Binding Sites
  • Catalytic Domain
  • Creatine / chemistry
  • Creatine / metabolism
  • Creatine Kinase / chemistry*
  • Creatine Kinase / genetics
  • Creatine Kinase / metabolism*
  • Creatine Kinase, Mitochondrial Form / chemistry*
  • Creatine Kinase, Mitochondrial Form / genetics
  • Creatine Kinase, Mitochondrial Form / metabolism*
  • Guanidine / chemistry
  • Humans
  • Magnesium / metabolism
  • Molecular Dynamics Simulation
  • Mutagenesis, Site-Directed

Substances

  • Adenosine Triphosphate
  • CKMT1B protein, human
  • Creatine Kinase
  • Creatine Kinase, Mitochondrial Form
  • Magnesium
  • Guanidine
  • Creatine