JBC, Vol. 250, Issue 20, 8193-8201, Oct, 1975
Magnetic resonance studies of the interaction of Co2+ and phosphoenolpyruvate with pyruvate kinase
E. Melamud and A. S. Mildvan
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.
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