Revelation of a catalytic calcium-binding site elucidates unusual metal dependence of a human apyrase

David W. Rooklin, Min Lu, Yingkai Zhang

Research output: Contribution to journalArticle

Abstract

Human soluble calcium-activated nucleotidase 1 (hSCAN-1) represents a new family of apyrase enzymes that catalyze the hydrolysis of nucleotide di- and triphosphates, thereby modulating extracellular purinergic and pyrimidinergic signaling. Among well-characterized phosphoryl transfer enzymes, hSCAN-1 is unique not only in its unusual calcium-dependent activation, but also in its novel phosphate-binding motif. Its catalytic site does not utilize backbone amide groups to bind phosphate, as in the common P-loop, but contains a large cluster of acidic ionizable side chains. By employing a state-of-the-art computational approach, we have revealed a previously uncharacterized catalytic calcium-binding site in hSCAN-1, which elucidates the unusual calcium-dependence of its apyrase activity. In a high-order coordination shell, the newly identified calcium ion organizes the active site residues to mediate nucleotide binding, to orient the nucleophilic water, and to facilitate the phosphoryl transfer reaction. From ab initio QM/MM molecular dynamics simulations with umbrella sampling, we have characterized a reverse protonation catalytic mechanism for hSCAN-1 and determined its free energy reaction profile. Our results are consistent with available experimental studies and provide new detailed insight into the structure-function relationship of this novel calcium-activated phosphoryl transfer enzyme.

Original languageEnglish (US)
Pages (from-to)15595-15603
Number of pages9
JournalJournal of the American Chemical Society
Volume134
Issue number37
DOIs
StatePublished - Sep 19 2012

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Apyrase
nucleotidase
Binding sites
Calcium
Metals
Binding Sites
Catalytic Domain
Enzymes
Nucleotides
Phosphates
Diphosphates
Molecular Dynamics Simulation
Amides
Hydrolysis
Ions
Protonation
human CANT1 protein
Water
Free energy
Molecular dynamics

ASJC Scopus subject areas

  • Chemistry(all)
  • Catalysis
  • Biochemistry
  • Colloid and Surface Chemistry

Cite this

Revelation of a catalytic calcium-binding site elucidates unusual metal dependence of a human apyrase. / Rooklin, David W.; Lu, Min; Zhang, Yingkai.

In: Journal of the American Chemical Society, Vol. 134, No. 37, 19.09.2012, p. 15595-15603.

Research output: Contribution to journalArticle

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