Determination of the screw sense specificity of bovine liver fructokinase.
Fructokinase from beef liver showed a clear reversal in specificity when the two isomers of ATP beta S were used as substrates with Mg2+ and Cd2+, with the Sp isomer having the higher V/K value with Mg2+ and the Rp isomer the higher value with Cd2+. The delta isomer of MgATP is thus the active form of the substrate. The substitution of sulfur for oxygen in the noncoordinated position of the beta-phosphate caused a 102-fold decrease in V/K over the value seen with MgATP, while substitution in the coordinated position gave a 21-fold decrease over the V/K value seen with CdATP. The Km values were little affected by sulfur substitution, showing that the wrong screw sense isomers were nonproductively bound almost as well as the correct ones. When ADP alpha S was used as a substrate in the reverse reaction, the Sp isomer showed the highest V/K value with both Mg2+ and Cd2+, suggesting that the metal ion is not coordinated to the alpha-phosphate during transphosphorylation. The failure of CrATP to act as a substrate for fructokinase suggests that the enzyme inserts one of its side chains into the inner coordination sphere of the metal ion during the reaction.
Pecoraro
V L
VL
Rendina
A R
AR
Cleland
W W
WW
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives; D000818:Animals; D001665:Binding Sites; D002104:Cadmium; D002417:Cattle; D005631:Fructokinases / Q000378:metabolism*; D066298:In Vitro Techniques; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D010770:Phosphotransferases / Q000378:metabolism*; D013237:Stereoisomerism; D013379:Substrate Specificity; D013873:Thionucleotides
D013873:Thionucleotides; D002104:Cadmium; C022570:adenosine 5'-O-(2-thiotriphosphate); D000255:Adenosine Triphosphate; D010770:Phosphotransferases; D005631:Fructokinases