The stereochemical configuration of Mn(II) . ADP at the active site of creatine kinase elucidated by electron paramagnetic resonance with Rp [alpha-17O]ADP and Sp [alpha-17O]ADP.

T. S. Leyh, R. D. Sammons, P. A. Frey, G. H. Reed

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Abstract

The stereochemical configuration of the Mn(II) . ADP complex at the active site of creatine kinase has been elucidated by EPR using chirally labeled [alpha-17O]ADP. Superhyperfine coupling between the 17O nucleus and Mn(II) produces a characteristic inhomogeneous broadening of the EPR signals for Mn(II) whenever the 17O is in the first coordination sphere of the metal ion. Previous experiments with ADP regio-specifically labeled with 17O in the phosphate groups had shown that Mn(II) was coordinated to the alpha and beta positions of ADP in transition state analog complexes with creatine kinase that involved enzyme, Mn(II), ADP, creatine, and anions such as formate and thiocyanate (Reed, G. H., and Leyh, T. S. (1980) Biochemistry 19, 5472-5480). The present experiments were initiated to determine the stereochemical configuration of the Mn(II) . ADP complex at the active site of the enzyme. EPR spectra for Mn(II) in transition state analog complexes with formate, thiocyanate, and nitrate as the stabilizing anions show inhomogeneous broadening from 17O in Sp [alpha-17O]ADP whereas the spectra obtained with Rp [alpha-17O]ADP are indistinguishable from those for matched samples with unlabeled ADP. The precision of the measurements indicates that the stereoselectivity of the enzyme for the delta configuration of the alpha, beta chelate of Mn(II) . ADP is greater than 15:1. The delta configuration is also preferred in fully active enzymic complexes involving the equilibrium mixture of substrates.

Original languageEnglish (US)
Pages (from-to)15047-15053
Number of pages7
JournalJournal of Biological Chemistry
Volume257
Issue number24
StatePublished - Dec 25 1982
Externally publishedYes

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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