Mechanistic and kinetics elucidation of Mg2+/ATP molar ratio effect on glycerol kinase

Getachew Shibabaw Molla, Alexander Himmelspach, Roland Wohlgemuth, Erhard T.K. Haupt, Andreas Liese

Research output: Contribution to journalJournal articleResearchpeer-review

Abstract

Detailed kinetics and mechanistic analyses of glycerol kinase from Cellulomonas sp. with respect to Mg2+ to ATP molar ratio were performed. The enzyme essentially requires Mg2+ for its activity and shows maximum activity at the optimum Mg2+ to ATP molar ratio of [0.12–0.3]. Subsequent increase of Mg2+ to ATP molar ratio higher than the values in the optimum region suppresses the enzyme activity to a non-zero asymptotic value. The enzyme exhibits two-step kinetics as a function of ATP at a fixed Mg2+ concentration due to the formation of multiple Mg-ATP complexes at different Mg2+ to ATP molar ratio. The addition of inorganic polyphosphate (PPin) inhibits or activates glycerol kinase due to the complexation of PPin with Mg2+ that shifts Mg2+ to ATP molar ratio below or to the optimum level. The change in all 31P NMR signals of ATP (i.e. α-, β- and γ-phosphate) by the addition of Mg2+ reveals that all of them are involved in Mg-ATP complex formation. The 1H NMR signals of CH2-protons on the ribose moiety of ATP become nearly equivalent after the addition of Mg2+ establishing the upper limit of optimum Mg2+ to ATP molar ratio for the enzyme activity. Therefore, it is concluded that the active site of glycerol kinase shows different catalytic property with respect to different Mg-ATP complexes. Glycerol kinase exhibits high affinity (low Km) and less activity (low kcat) for complexes with a stoichiometric or over-stoichiometric constitution like Mg2ATP. On the other hand, the enzyme shows less affinity (high Km) and high activity (high kcat) for unsaturated complexes like [Mg(ATP)2]−6.
Original languageEnglish
JournalJournal of Molecular Catalysis
Volume445
Pages (from-to)36-42
DOIs
Publication statusPublished - 2018
Externally publishedYes

Keywords

  • Glycerol kinase kinetics
  • Mg2+ to ATP ratio
  • Two-step enzyme kinetics
  • Mg-ATP complexes
  • 31P NMR

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