Magnetic resonance studies of the interaction of Co2+ and phosphoenolpyruvate with pyruvate kinase.
Co2+, which activates rabbit muscle pyruvate kinase, competes with Mn2+ for the active site of the enzyme with a KD of 46 muM. Co2+ binds to phosphoenolpyruvate with a KD of 4.1 mM. The structures of the binary Co2+/P-enolpyruvate, and quaternary pyruvate kinase/Co2+/K+/P-enolpyruvate complexes were studied using EPR and the effects of Co2+ on the longitudinal (T1) and transverse (T2) relaxation times of the protons of water and P-enolpyruvate and the phosphorus of P-enolpyruvate. The EPR spectra of all complexes at 6 K, disappear above 40 K and reveal principal g values between 2 and 7 indicating high spin Co2+. For free Co2+ and for the binary Co2+/P-enolpyruvate complex, the T1 of water protons was independent of frequency in the range 8, 15, 24.3, 100, and 220 MHz. Assuming coordination numbers (q) of 6 and 5 for free Co2+ and Co2+/P-enolpyruvate, respectively, correlation times (tauc) of 1.3 times 10(-13) and 1.7 times 10(-13) s, were calculated. The distances from Co2+ and phosphorus and to the cis and trans protons in the binary Co2+/P-enolpyruvate complex calculated from their T1 values were 2.7 A, 4.1 A, AND 5.3 A, respectively, indicating an inner sphere phosphoryl complex. Consistent with direct phosphoryl coordination, a large Co2+ to phosphorus hyperfine contact coupling constant (A/h) of 5 times 10(5) Hz was determined by the frequency dependence of the T2 of phosphorus at 25.1, 40.5, and 101.5 MHz. For both enzyme complexes, the dipolar correlation time tauc was 2 times 10(-12) s and the number of rapidly exchanging water ligands (q) was 0.6 as determined from the frequency dependence of the T1 of water protons. In the quaternary enzyme/Co2+/K+/P-enolyruvate complex this tauc value was consistent with the frequency dependence of the T1 of the phosphorus of enzyme-bound P-enolpyruvate at 25.1 and 40.5 MHz. Distances from enzyme-bound C02+ to the phosphorus and protons of P-enolpyruvate, from their T1 values, were 5.0 A and 8 to 10 A, respectively, indicating a predominantly (greater than or equal to 98%) second spere complex and less than 2% inner sphere complex. Consistent with a second sphere complex on the enzyme, an A/h value of less than 10(3) Hz was determined from the frequency dependence of the T2 of phosphorus. In all complexes the exchange reates were found to be faster than the paramagnetic relaxation rates and the hyperfine contact interaction was found to be small compared to the dipolar interaction. The results thus indicate that the interaction of C02+ with P-enolpyruvate is greatly decreased upon binding to the active site of pyruvate kinase.
D000818:Animals; D001665:Binding Sites; D003035:Cobalt / Q000494:pharmacology*; D004789:Enzyme Activation / Q000187:drug effects; D009682:Magnetic Resonance Spectroscopy; D008345:Manganese / Q000494:pharmacology; D008433:Mathematics; D008958:Models, Molecular; D009132:Muscles / Q000201:enzymology; D010728:Phosphoenolpyruvate / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D011770:Pyruvate Kinase / Q000378:metabolism*; D011817:Rabbits
D003035:Cobalt; D008345:Manganese; D010728:Phosphoenolpyruvate; D011770:Pyruvate Kinase