Dynamic coordination of two-metal-ions orchestrates λ-exonuclease catalysis

• Published in: Nature communications Vol. 9; no. 1; pp. 4404 - 10
• Main Authors: Hwang, Wonseok, Yoo, Jungmin, Lee, Yuno, Park, Suyeon, Hoang, Phuong Lien, Cho, HyeokJin, Yu, Jeongmin, Hoa Vo, Thi Minh, Shin, Minsang, Jin, Mi Sun, Park, Daeho, Hyeon, Changbong, Lee, Gwangrog
• Format: Journal Article
• Language: English
• Published: England Nature Publishing Group 23.10.2018; Nature Publishing Group UK; Nature Portfolio
• Subjects: Binding sites; Biocatalysis; Borides; Calcium - pharmacology; Catalysis; Cofactors; Degradation; Deoxyribonucleic acid; DNA; DNA - metabolism; Enzymatic activity; Enzyme activity; Enzymes; Exonuclease; Exonucleases - metabolism; Fluorescence; Fluorescence Resonance Energy Transfer; Heterogeneity; Ions; Kinetics; Magnesium; Magnesium - pharmacology; Magnesium compounds; Metal ions; Metals - chemistry; Models, Molecular; Single Molecule Imaging; Translocation; Variations
• ISSN: 2041-1723; 2041-1723
• DOI: 10.1038/s41467-018-06750-9

Metal ions at the active site of an enzyme act as cofactors, and their dynamic fluctuations can potentially influence enzyme activity. Here, we use λ-exonuclease as a model enzyme with two Mg binding sites and probe activity at various concentrations of magnesium by single-molecule-FRET. We find that while Mg and Mg have similar binding constants, the dissociation rate of Mg is two order of magnitude lower than that of Mg due to a kinetic-barrier-difference. At physiological Mg concentration, the Mg ion near the 5'-terminal side of the scissile phosphate dissociates each-round of degradation, facilitating a series of DNA cleavages via fast product-release concomitant with enzyme-translocation. At a low magnesium concentration, occasional dissociation and slow re-coordination of Mg result in pauses during processive degradation. Our study highlights the importance of metal-ion-coordination dynamics in correlation with the enzymatic reaction-steps, and offers insights into the origin of dynamic heterogeneity in enzymatic catalysis.