Brain hexokinase has no preexisting allosteric site for glucose 6-phosphate.
Difference spectroscopic investigations on the interaction of brain hexokinase with glucose and glucose 6-phosphate (Glc-6-P) show that the binary complexes E-glucose and E-Glc-6-P give very similar UV difference spectra. However, the spectrum of the ternary E-glucose-Glc-6-P complex differs markedly from the spectra of the binary complexes, but resembles that produced by the E-glucose-Pi complex. Direct binding studies of the interaction of Glc-6-P with brain hexokinase detect only a single high-affinity binding site for Glc-6-P (KD = 2.8 microM). In the ternary E-glucose-Glc-6-P complex, Glc-6-P has a much higher affinity for the enzyme (KD = 0.9 microM) and a single binding site. Ribose 5-phosphate displaces Glc-6-P from E-glucose-Glc-6-P only, but not from E-Glc-6-P complex. It also fails to displace glucose from E-glucose and E-glucose-Glc-6-P complexes. Scatchard plots of the binding of glucose to brain hexokinase reveal only a single binding site but show distinct evidence of positive cooperativity, which is abolished by Glc-6-P and Pi. These ligands, as well as ribose 5-phosphate, substantially increase the binding affinity of glucose for the enzyme. The spectral evidence, as well as the interactive nature of the sites binding glucose and phosphate-bearing ligands, lead us to conclude that an allosteric site for Glc-6-P of physiological relevance occurs on the enzyme only in the presence of glucose, as a common locus where Glc-6-P, Pi, and ribose 5-phosphate bind. In the absence of glucose, Glc-6-P binds to the enzyme at its active site with high affinity. We also discuss the possibility that, in the absence of glucose, Glc-6-P may still bind to the allosteric site, but with very low affinity, as has been observed in studies on the reverse hexokinase reaction.
Mehta
A
A
Tata Institute of Fundamental Research, Bombay, India.
Jarori
G K
GK
Kenkare
U W
UW
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D001921:Brain / Q000201:enzymology*; D002417:Cattle; D019298:Glucose-6-Phosphate; D005958:Glucosephosphates / Q000378:metabolism*; D006593:Hexokinase / Q000378:metabolism*; D007700:Kinetics; D012267:Ribosemonophosphates / Q000378:metabolism; D013056:Spectrophotometry, Ultraviolet
D005958:Glucosephosphates; D012267:Ribosemonophosphates; C031626:ribose-5-phosphate; D019298:Glucose-6-Phosphate; D000255:Adenosine Triphosphate; D006593:Hexokinase