Yeast phosphofructokinase: studies on thiol reactivity of a cross-linked enzyme form.
Cross-linked yeast phosphofructokinase exhibiting the basic regulatory properties of the unmodified enzyme but no cooperativity with respect to the substrate fructose 6-phosphate was subjected to stopped-flow thiol titration in order to study the influence of allosteric effectors on the level of protein conformation. As found for native phosphofructokinase, the cross-linked enzyme revealed at least two classes of cysteinyl residues which can be distinguished by their reactivity towards 5,5'-dithiobis-(2-nitrobenzoic acid). In spite of the restricted conformational flexibility caused by the intramolecular cross-linking, several fructose phosphates and AMP were capable of diminishing the apparent first-order rate constant (k) of modification of the fast reacting thiol groups by Ellman's reagent. In the presence of ATP, a drastic decrease of the k-value by more than one order of magnitude became apparent. The data are appropriate to support the hypothesis of the existence of multiple conformational determinants in octameric yeast phosphofructokinase and contribute to the understanding of the extremely different stability of the enzyme in the presence of either fructose 6-phosphate or ATP with respect to proteolytic degradation.
Kriegel
T
T
Institut für Biochemie, Universität Leipzig, FRG.
Bär
J
J
Hübner
G
G
Kopperschläger
G
G
D000255:Adenosine Triphosphate / Q000378:metabolism; D003432:Cross-Linking Reagents; D004228:Dithionitrobenzoic Acid / Q000378:metabolism; D005635:Fructosediphosphates / Q000378:metabolism; D005636:Fructosephosphates / Q000378:metabolism; D005656:Fungal Proteins / Q000378:metabolism*; D010732:Phosphofructokinase-1 / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000201:enzymology*
D003432:Cross-Linking Reagents; D005635:Fructosediphosphates; D005636:Fructosephosphates; D005656:Fungal Proteins; C027618:fructose-6-phosphate; C027652:fructose 2,6-diphosphate; D000255:Adenosine Triphosphate; D004228:Dithionitrobenzoic Acid; D010732:Phosphofructokinase-1