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 ϳ100 mg/liter. The steady-state kinetic parameters for both forward and reverse reactions were determined by initial velocity measurements. The turnover numbers (k cat ) 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). K m 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 kinase⅐MgADP⅐GMP. 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.
Guanylate kinase (GK)1 catalyzes the reversible phosphoryl transfer from ATP to GMP in the presence of Mg 2ϩ (1). The enzyme is essential for converting GMP to GDP and therefore synthesis of GTP. It plays an important role in the cGMP cycle (2) and is also required for metabolic activation of the antiviral drugs acyclovir and gancyclovir (3, 4).GK has been purified to various degrees of purity from several sources (5 and references therein), but detailed characterization has been hampered by its low abundance. Mammalian GK has been characterized by steady-state kinetics (1, 2), but no further mechanistic studies have been reported. It was not until 1989 that yeast GK was purified to homogeneity, and its amino acid sequence was determined by peptide sequencing (6). The crystal structure of yeast GK in complex with GMP was solved and refined shortly afterwards at 2-Å resolution (7, 8).The genes for yeast GK, Escherichia coli GK, and bovine GK were recently cloned (9 -11). The amino acid sequence of porcine brain GK was determined by peptide sequencing (5). Yeast GK shares 44.8% identity with E. coli GK, 51% identity with porcine GK, and 55% identity with bovine GK. It is noteworthy that, unlike the yeast and mammalian GKs, which are monomeric, E. coli GK is tetrameric at low salt conditions and dimeric at high salt conditions (10). Interestingly, several proteins, including the protein encoded by Drosophila tumor suppresser gene dlg-A (12), a rat presynaptic density protein (SAP90) (13), a rat postsynaptic density protein (PSD-95) (14), and the major pa...