Substrate synergism and the kinetic mechanism of yeast hexokinase.
Michaelis constants for MgATP with yeast hexokinase vary from 28 microM with D-mannose to above 4 mM for the slow ATPase reaction, with the different values reflecting the degree of synergism in binding of MgATP and the sugar substrate. The best substrates show the greatest synergism, but the correlation is not exact. Similar synergistic binding between MgADP or its methylene analogue and phosphorylated sugars is seen. Product inhibiton of MgADP vs. MgATP and vice versa appears noncompetitive at low levels of variable substrate but becomes competitive at high levels. These patterns show that MgATP can combine with E-glucose-6-P (Ki = 4 mM) and MgADP with E-glucose (Ki = 1.6 mM). Isotope partitioning studies with glucose or glucose-6-P have determined the rates of release of these substrates from binary and ternary complexes and, together with reverse isotope exchange studies and the product inhibition studies mentioned above, have shown that the kinetic mechanism is a somewhat random one in which dissociation of sugars from productive ternary complexes is very slow, but release from nonproductive ternary complexes occurs at rates similar to those from binary enzyme-sugar complexes. D-Arabinose-5-P has a Km of 4.6 mM and a Vmax 5% that for glucose-6-P, confirming that the high Km for D-arabinose in the forward direction is caused by the low proportion in the furanose form. The dissociation constant of MgADP in the absence of sugars was determined from the Ki of 5.8 mM for MgADP as a competitive inhibitor vs. MgATP of the slow ATPase reaction.
D000244:Adenosine Diphosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D001667:Binding, Competitive; D002384:Catalysis; D005947:Glucose / Q000378:metabolism; D006593:Hexokinase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D007700:Kinetics; D008956:Models, Chemical; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D013379:Substrate Specificity
D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D006593:Hexokinase; D005947:Glucose