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

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 /T l ) 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 measured distances in the enzyme-Mn2+-M+-phosphoenolpyruvate (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 disA 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 activities 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 activation 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 monovalent 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 enzymeMn2+-T1+-phosphoenolpyruvate (PEP) complex and the enzyme-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 From the Department of Chemistry, The Pennsylvania State University, University Park, Pennsylvania 16802. Received April 8, 1980. Supported by the National Science Foundation (PCM-7807845). Support for the nuclear magnetic resonance instrumentation from the National Science Foundation is also gratefully acknowledged. *Correspondence should be addressed to this author. J.J.V. is an Established Investigator of the American Heart Association. * F.M.R. is a National Research Service awardee (AM-05996). Present address: Department of Chemistry, Texas A&M University, College Station, TX 77843. tances 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 (rC) for the dipolar Mn2+-M+ interaction. From the ratio of the paramagnetic effects of enzyme-Mn2+ on the l / T I values of the two isotopes of Li+, NH4+, or Rb+, a unique value of T~ is obtained at a single magnetic field strength. Further analysis of this method reveals that for 6Li+, 7Li+, 15NH4+, and 133Cs+, distances from enzyme-Mn2+ to these monovalent cations can be reliably measured up to 12-20 A, while for 39K+, s5Rb+, and 87Rb+ the practical upper limit is -4 A. For 14NH4+, 205Tl+, and 23Na+, 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. only 3% as well as T1+, these authors proposed that the longer metal-metal distance for the Li+ complexes compared with the T1+ complexes correlated with the large decrease in enzymatic activity. Ash et al. (1978) pointed out that there are two interconvertible forms of the enzyme-Mn2+-Li+-PEP complex (Reed & Cohn, 1973) that Hutton et al. (1977) did not take account of in their data analysis. Repeating the 7Li NMR experiments, Ash et al. (1 978) obtained distances between Mn2+ and Li+ that are nearly identical with the MnZ+-Tl+ distances of Reuben dt Kayne (1 97 1). However, the reason for the difference in the measured distances in these two 7Li NMR studies is in the choice of a correlation time (TJ for the dipolar Mn2+-Li+ interaction. Hutton et al. (1977) used a value of 9.4 ns while Ash et al. (1978) used a value of 1.7 ns. These apparently disparate correlation times were obtained from a study of water proton relaxation rates. Clarification of this discrepancy is needed. In this paper we report an extended study of the monovalent and divalent cation sites of pyruvate kinase which includes most of the monovalent cations that activate pyruvate kinase. The ions investigated were 6Li+, 7Li+, 14NH 4 , + 15NH4+, 23Na+, 39K+, 85Rb+, 87Rb+, and 133Cs+. Although most of these isotopes are quadrupolar and some have very fast relaxation rates, we previously pointed out that a number of them are particularly attractive for study by NMR (Villafranca & Raushel, 1980). Additionally, we developed a novel method for unambiguously determining the correlation time for the dipolar interaction of a paramagnetic center with monovalent cations (Raushel & Villafranca, 1980). This method is based on determining the ratio of spin-lattice relaxation rates of the two isotopes of Li+, NH4+, or Rb+ interacting with an enzyme-bound paramagnetic center, e.g., Mn2+. The calculated ' Abbreviations used: NMR, nuclear magnetic resonance; PEP, phosphoenolpyruvate; EDTA, ethylenediaminetetraacetic acid; EPR, electron paramagnetic resonance.