Cooperative effect of fructose bisphosphate and glyceraldehyde-3-phosphate dehydrogenase on aldolase action.
The combination of binding and kinetic approaches is suggested to study (i) the mechanism of substrate-modulated dynamic enzyme associations; (ii) the specificity of enzyme interactions. The effect of complex formation between aldolase and glyceraldehyde-3-phosphate dehydrogenase (D-glyceraldehyde-3-phosphate:NAD+ oxidoreductase (phosphorylating), EC 1.2.1.12) on aldolase catalysis was investigated under pseudo-first-order conditions. No change in kcat but a significant increase in KM of fructose 1,6-bisphosphate for aldolase was found when both enzymes were obtained from muscle. In contrast, kcat rather than KM changed if dehydrogenase was isolated from yeast. Next, the conversion of fructose 1-phosphate was not affected by interactions between enzyme couples isolated from muscle. The influence of fructose phosphates on the enzyme-complex formation was studied by means of covalently attached fluorescent probe. We found that the interaction ws not perturbed by the presence of fructose 1-phosphate; however, fructose 1,6-bisphosphate altered the dissociation constant of the enzyme complex. A molecular model for fructose 1,6-bisphosphate-modulated enzyme interaction has been evaluated which suggests that high levels of fructose bisphosphate would drive the formation of the 'channelling' complex between aldolase and glyceraldehyde-3-phosphate dehydrogenase.
Neuzil
J
J
Institute of Enzymology, Hungarian Academy of Sciences, Budapest.
Danielson
H
H
Welch
G R
GR
Ovádi
J
J
D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive; D016650:Fluorescein-5-isothiocyanate; D005452:Fluoresceins; D005456:Fluorescent Dyes; D005634:Fructose-Bisphosphate Aldolase / Q000378:metabolism*; D005635:Fructosediphosphates / Q000378:metabolism / Q000494:pharmacology*; D005636:Fructosephosphates / Q000378:metabolism; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000378:metabolism*; D006598:Hexosediphosphates / Q000494:pharmacology*; D007700:Kinetics; D009132:Muscles / Q000201:enzymology; D011092:Polyethylene Glycols / Q000494:pharmacology; D011817:Rabbits; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D013045:Species Specificity; D013050:Spectrometry, Fluorescence; D013861:Thiocyanates
D005452:Fluoresceins; D005456:Fluorescent Dyes; D005635:Fructosediphosphates; D005636:Fructosephosphates; D006598:Hexosediphosphates; D013861:Thiocyanates; C032284:fructose-1-phosphate; D011092:Polyethylene Glycols; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; D005634:Fructose-Bisphosphate Aldolase; D016650:Fluorescein-5-isothiocyanate; C029063:fructose-1,6-diphosphate