Phosphorus-31 nuclear relaxation rate studies of the nucleotides on phosphoenolpyruvate carboxykinase

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Abstract
The interactions of nucleotide substrates with the enzyme phosphoenolpyruvate carboxykinase and its Mn2+ complex were investigated by several methods. Direct binding shows the formation of stoichiometric complexes. The presence of Mn2+ increases the affinity of the enzyme for nucleotide. A higher affinity for GTP (Kd < 2 .mu.M) than for GDP (Kd = 15 .mu.M) was determined. Solvent proton relaxation rate studies indicate no substantial difference in titration curves for free nucleotide or for Mg-nucleotide to the enzyme-Mn complex. The effect of Mn2+ on the 31P relaxation rates of IDP and of ITP in the binary Mn-nucleotide complex indicates the formation of direct coordination complexes. The distances of the .alpha.- and .beta.-31P of IDP to Mn2+ are identical (3.5 .ANG.). The Mn2+ distance to the .beta.- and .gamma.-31P of ITP is also identical (3.7 .ANG.) and is 0.2 .ANG. further from the .alpha.-phosphorus. In the presence of phosphoenolpyruvate carboxykinase, the effect of Mn2+ on the 31P relaxation rates was measured at 40.5 MHz and at 121.5 MHz. The dipolar correlation time was calculated to be 0.6-5.4 ns, depending upon assumptions made. The Mn2+ to phosphorus distances indicate the nucleotide substrates form a 2nd sphere complex to the bound Mn2+. From 1/T2 measurements, electron delocalization from Mn2+ to the P atoms is indicated; this effect occurs although direct coordination does not take place. The exchange rate of GTP from the enzyme-Mn complex (koff = 4 .times. 104 s-1) is rapid compared to kcat with a lower energy of activation (9.2 kcal/mol) than for catalytic turnover. The bound Mn2+ must exert electronic effects across long distances at the catalytic site.