In order to compare with hGMPK, the gene gmk has been cloned and the protein over-expressed and purified. Prior to this work,
LBH 589 of hGMPK in E. coli or production by cell-free translations from the human gmk gene resulted in inactive enzyme [24]. In a GST expression vector, the enzyme has been characterized by nanoelectrospray mass spectrometry, but without kinetic analysis [25]. Here recombinant His-tagged hGMPK was successfully expressed in E. coli and highly purified to a final concentration of 30 mg/L of cell culture medium (Fig. S1). In the presence of ATP, hGMPK accepted GMP as a substrate with parameters (Km = 0.05 mM, kcat = 500 s?1, kcat/Km = 107 M?1s?1) that were close to those of dGMP ( Table 1). No evidence of inhibition by high concentrations of GMP was observed (result not shown), in
contrast to the yeast enzyme [26]. Our data are in agreement with the properties of hGMPK purified from human erythrocytes [27] and [28]. Additionally, hGMPK was found unable to phosphorylate TMP ( Fig. 3).
