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

Quanjie Li,Yuanyuan Jin,Jinming Zhou,Xiaoyu Li,Shuai Fan,Weiqing He,Zhaoyong Yang,Shan Cen

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.

Highlights

CKs share ~60% sequence identity across all species and among the isoforms[9]
Our study on the phosphoryl transfer reaction of human ubiquitous mitochondrial creatine kinase (uMtCK) gives new insights into its catalytic mechanism by biochemical assay coupled with theoretically molecular dynamic (MD) simulation and quantum chemistry calculation
Because the crystal structure of uMtCK represented the unliganded “open” state, we predicted a 3D model of uMtCK in complex with the co-factor ATP-Mg2+ and creatine

Summary

Introduction

In the CK family including human uMtCK10, 13 crystal structures have been reported and can be sorted into three groups according to the substrate binding mode: the ligand-free-form[11,12,13,14], the ADP-Mg2+-complex[15,16], and the ADP-Mg2+·nitrate·creatine transition-state analogue complex[17,18,19]. Milner-White and Watts firstly proposed that CK forms a quaternary CK·ADP-Mg2+·nitrate·creatine complex, in which the nitrate mimics the planar γ-phosphate in the transition state[28]. This was subsequently confirmed by the transition-state analogue complex structures of human BB-CK (PDB entry:3B6R)[17], rabbit MM-CK (PDB entry:1U6R)[19], and torpedo california MM-CK (PDB entry:1VRP)[18]. Our study on the phosphoryl transfer reaction of human uMtCK gives new insights into its catalytic mechanism by biochemical assay coupled with theoretically MD simulation and quantum chemistry calculation

Methods

Results

Conclusion