31P NMR of enzyme-bound substrates of rabbit muscle creatine kinase. Equilibrium constants, interconversion rates, and NMR parameters of enzyme-bound complexes.
The reaction catalyzed by rabbit muscle creatine kinase ATP + creatine in equilibrium ADP + P-creatine has been investigated by 31P NMR. At pH 8.0 and 4 degrees C, the equilibrium constant of the overall reaction [P1][P2]/[S1] [S2] is found to be 0.08, while that for the interconversion step between enzyme-bound substrates and products [E.P1. P2]/[E.S1.S2] is estimated to be approximately 1; the latter value is the same for all other kinases investigated. The rate of interconversion of enzyme-bound substrates and products is approximately 90 s-1 and is not the rate-limiting step of the overall reaction. Of the phosphate groups in enzyme complexes of reactants or products, the 31P chemical shifts of beta-P(ADP) and beta-P[MgADP) change by approximately 2 ppm downfield while all others change by less than 0.8 ppm. In the transition state analog complexes E.MgADP.NO3-.creatine and E.MgADP.HCOO-.creatine, the beta-P(MgADP) signal shows a substantial upfield shift in the direction of the beta-P(MgATP) resonance. The pattern of chemical shifts and line shapes of nucleotide complexes of creatine kinase parallel those for the corresponding complexes of arginine kinase, indicating structural and/or conformational similarity of the phosphate chains of nucleotides bound to the two enzymes. However, a difference in active sites is indicated by the pH independence (pH 6.0 to 9.0) of the chemical shift of the beta-P of MgADP bound to creatine kinase, whereas with arginine kinase this resonance showed a pKa approximately 7.5.
D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D000818:Animals; D003401:Creatine; D003402:Creatine Kinase / Q000378:metabolism*; D007700:Kinetics; D008274:Magnesium; D009682:Magnetic Resonance Spectroscopy; D009132:Muscles / Q000201:enzymology*; D011485:Protein Binding; D011817:Rabbits
D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D003402:Creatine Kinase; D008274:Magnesium; D003401:Creatine