Kinetic and thermodynamic characterizations of yeast guanylate kinase.
Yeast guanylate kinase was expressed at high level in Escherichia coli using pET-17b vector. It was purified to homogeneity by a simple two-column procedure with an average yield of approximately 100 mg/liter. The steady-state kinetic parameters for both forward and reverse reactions were determined by initial velocity measurements. The turnover numbers (kcat) were 394 s-1 for the forward reaction (formation of ADP and GDP) and 90 s-1 for the reverse reaction (formation of ATP and GMP). Km values were 0.20, 0. 091, 0.017, and 0.097 mM for MgATP, GMP, MgADP, and GDP, respectively. Analysis of the initial velocity patterns indicated a sequential mechanism. GMP was found to have partial substrate inhibition. The substrate inhibition was not competitive with MgATP and could be attributed to formation of the abortive complex guanylate kinase.MgADP.GMP. The equilibrium constant of the reaction was measured under various conditions by NMR and a radiometric assay. The results showed that the steady-state kinetic parameters were consistent with the thermodynamic constant. NMR titration and equilibrium dialysis showed that both substrates and products could bind to free guanylate kinase. The dissociation constants were 0.090, 0.18, 0.029, 0.084, and 0.12 mM for MgATP, ATP, GMP, MgADP, and GDP, respectively. Viscosity-dependent kinetics was used to identify the rate-limiting steps of the reaction. The results indicated that the reaction rate is largely controlled by the chemical step.
Li
Y
Y
Department of Biochemistry, Michigan State University, East Lansing, Michigan 48824, USA.
Zhang
Y
Y
Yan
H
H
D000244:Adenosine Diphosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D006157:Guanosine Monophosphate / Q000378:metabolism; D051528:Guanylate Kinases; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D009703:Nucleoside-Phosphate Kinase / Q000378:metabolism*; D013816:Thermodynamics; D014783:Viscosity
D000244:Adenosine Diphosphate; D006157:Guanosine Monophosphate; D000255:Adenosine Triphosphate; D009703:Nucleoside-Phosphate Kinase; D051528:Guanylate Kinases