Nuclear Magnetic Resonance Study of the Binding of Phosphoenolpyruvate and Phosphoenol-α-ketobutyrate to Manganese Pyruvate Kinase: TEMPERATURE, FREQUENCY, AND MONOVALENT CATION DEPENDENCE OF WATER PROTON NUCLEAR MAGNETIC RESONANCE RELAXATION RATES

Abstract

Abstract The proton magnetic resonance relaxation rate (PRR) of water in ternary complexes of pyruvate kinase-Mn(II) with P-enolpyruvate and P-enol-α-ketobutyrate has been examined in the presence of various monovalent cations by titration of pyruvate kinase-Mn(II) with either P-enolpyruvate or P-enol-α-ketobutyrate. The calculated dissociation constants of the substrate and of the enzyme from the enzyme-metal-substrate complexes, K3 and K2 respectively, and the enhancement of the ternary P-enolpyruvate complex (et) were found to depend upon the monovalent cation present. The results are interpreted in terms of an equilibrium mixture of the two known conformational forms of the ternary P-enolpyruvate complex in the presence of nonactivating (CH3)4N+. In the presence of activating K+, only one ternary species exists which is the same as the major conformational species formed with (CH3)4N+. By the EPR criterion, the substrate analog P-enol-α-ketobutyrate forms only one species of the enzyme-Mn(II)-substrate complex in the presence of either (CH3)4N+ or K+ and, unlike the P-enol-pyruvate complex, the value of et for the analog complex is independent of monovalent cation. It is concluded that the monovalent cation affects only the equilibrium between the two conformational species of the P-enolpyruvate complex and that the enhancement of the major conformational form is invariant with monovalent cation. From the fact that the PRR enhancement et of the minor conformational form (∼17) is much closer to that of the binary metal-enzyme complex (eb = 22.5) than to that of the ternary K+ complex (et = 2.3), it is inferred that the Mn(II) active site environment of the minor conformational form closely resembles that of the binary complex. PRR titrations for the P-enolpyruvate complex have yielded a stoichiometry of 3.5 P-enolpyruvate sites per molecule of pyruvate kinase. Temperature and frequency dependence studies of the water PRR for the ternary P-enolpyruvate complex in the presence of K+ bracketed the correlation time for the dipolar interaction between Mn(II) and the water protons: 0.7 x 10-9 s ≤ τc l 2.6 x 10-9 s. In the presence of (CH3)4N+, the water PRR for the ternary P-enolpyruvate complex is frequency-dependent. The frequency-dependent component is identified with the minor conformational species present only in (CH3)4N+ solutions. The number, n, of water molecules in the first coordination sphere of Mn(II) of the ternary P-enolpyruvate complex in the presence of K+ was calculated to be 0.2 ≤ n ≤ 0.5. The low value of n is discussed in terms of possible structural features of the enzyme-metal-substrate complex.