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

Abstract

The effectiveness of measuring distances between monovalent and divalent cation sites on enzymes has been examined by 6Li, 7Li, I4N, 15N, 23Na, 39K, 85Rb, 87Rb, and 133Cs nuclear magnetic resonance (NMR). Measurements were made of the paramagnetic effect of enzyme-bound Mn2+ on the longitudinal spin-lattice relaxation rate ( l/Tl) of the monovalent cations by using MnZ+ at the divalent cation site of pyruvate kinase. Distances from MnZ+ 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 mea- sured distances in the enzyme-Mn2+-M+-phosphoenol- pyruvate (PEP) complex are as follows: 6Li+, 5.7 A; 7Li+, 5.7 A; I4NH4+, 4.4 A; 15NH4+, 4.4 A; 133Cs+, 6.0 A. In the complex with PEP, a lower limit distance could be placed on MnZ+ to 23Na+ (24.5 A), 39K+ (23.7 A), and 87Rb+ (24.1 A). These results show a 2-3-A reduction in the distance between MnZ+ and the monovalent cation upon addition of PEP to the enzyme. Additionally, the MnZ+ to monovalent cation dis- A large number of enzymes display an absolute requirement for the addition of a monovalent cation for maximal activity (Suelter, 1970). The most studied enzyme of this group is pyruvate kinase which shows a wide range of maximal ac- tivities depending on the monovalent cation used to activate the enzyme (Kayne, 1973). For example, lithium activates only 2% as well as does potassium. The other monovalent cations are of intermediate activity (Kayne, 1973). It has been observed that the degree of activation of pyruvate kinase by the various monovalent cations appears to correlate with the crystalline ionic radius of these cations. The amount of ac- tivation is found to decrease as the ionic radius increases or decreases relative to that for K+ (Kachmar & Boyer, 1953). However, the precise function of these monovalent cations in catalysis is not known. In an attempt to determine the exact location of the mo- novalent cation site relative to the sites for the other ligands of pyruvate kinase, a number of laboratories have undertaken measurements of the distance from enzyme-bound Mn2+ to the monovalent cation site by using nuclear magnetic resonance (NMR).' Reuben & Kayne (1971) have reported distances of 4.9 and 8.2 A between 205Tl+ and Mn2+ in the enzyme- Mn2+-T1+-phosphoenolpyruvate (PEP) complex and the en- zyme-Mn2+-T1+ complex, respectively. In a 7Li NMR study, Hutton et al. (1977) reported distances of 5.8 and 11 .O A for these enzyme-Mn2+ complexes with 7Li+. Since Li+ activates