A multinuclear nuclear magnetic resonance study of the monovalent-divalent cation sites of pyruvate kinase. Academic Article uri icon


  • The effectiveness of measuring distances between monovalent and divalent cation sites on enzymes has been examined by6Li,7Li,14N,15N,23Na,39K,85Rb,87Rb, and133Cs nuclear magnetic resonance (NMR). Measurements were made of the paramagnetic effect of enzyme-bound Mn2+on the longitudinal spin-lattice relaxation rate (1/T1) of the monovalent cations by using Mn2+at the divalent cation site of pyruvate kinase. Distances from Mn2+to the monovalent cations in the enzyme-Mn2+-M+complex are as follows:6Li+, 8.5 A;7Li+, 8.4 A;15NH5+7.0 A;133Cs+7.7 A. The measured distances in the enzyme-Mn2+-M+-phosphoenolpyruvate (PEP) complex are as follows:6Li+, 5.7 A;7Li+, 5.7 A;14NH4+, 4.4 A;15NH4+, 4.4 A;l33Cs+, 6.0 A. In the complex with PEP, a lower limit distance could be placed on Mn2+to23Na+(4.5 A),39K+(3.7 A), and87Rb+(4.1 A). These results show a 2-3-A reduction in the distance between Mn2+and the monovalent cation upon addition of PEP to the enzyme. Additionally, the Mn2+ to monovalent cation distances in the enzyme complexes with PEP correlate very well with the observed levels of activation of pyruvate kinase by these ions. In this study we used a novel method to determine the correlation time (c) for the dipolar Mn2+-M+interaction. From the ratio of the paramagnetic effects of enzyme-Mn2+on the 1/T1) values of the two isotopes of Li+, NH4+, or Rb+, a unique value of cis obtained at a single magnetic field strength. Further analysis of this method reveals that for6Li+,7Li+,15NH4+, and133Cs+, distances from enzyme-Mn2+to these monovalent cations can be reliably measured up to 12-20 A, while for39K+,85Rb+, and87Rb+the practical upper limit is ~4 A. For14NH4+,205Tl+, and23Na+Mn2+to M+distances could be measured in the 5-8-A range. Thus, the multinuclear NMR study of the monovalent cation site of pyruvate kinase reported herein has revealed the feasibility of using NMR to relate structure to function in the large class of enzymes that are activated by monovalent cations. 1980, American Chemical Society. All rights reserved.

published proceedings

  • Biochemistry

author list (cited authors)

  • Raushel, F. M., & Villafranca, J. J.

citation count

  • 34

complete list of authors

  • Raushel, FM||Villafranca, JJ

publication date

  • November 1980