Functional organization of mammalian hexokinases: characterization of chimeric hexokinases constructed from the N- and C-terminal domains of the rat type I and type II isozymes.
Chimeric hexokinases consisting of either the N-terminal half of Type I hexokinase fused with the C-terminal half of the Type II isozyme (NICII) or the inverse pair (NIICI), along with the parental isozymes, were expressed in COS-1 cells. The thermal stability of the chimeras was intermediate between that of the highly labile Type II isozyme and the relatively stable Type I hexokinase. In their Kms for substrates, Glc and ATP, the chimeric enzymes were similar to the parental isozyme from which the C-terminal half was derived. Although the Type I and Type II isozymes were similar in their sensitivity to inhibition (competitive vs ATP) by the Glc-6-P analogs, 1,5-anhydroglucitol 6-phosphate (AnGlc-6-P), and Glc-1,6-bisphosphate, the chimeric enzymes differed markedly, with the NIICI chimera being much more sensitive and the NICII chimera much less sensitive than either parental form to these inhibitors. In contrast, the response of the chimeras to Pi, either as an antagonist of inhibition by AnGlc-6-P or, at higher concentrations, as an inhibitor, was correlated with the origin of the N-terminal domain. The results are consistent with the view that catalytic function is associated with the C-terminal domain of the Type I isozyme, with regulatory function--inhibition by Glc-6-P and its analogs and antagonism of this inhibition by Pi--being mediated by the N-terminal domain.
Tsai
H J
HJ
Department of Biochemistry, Michigan State University, East Lansing 48824.
Wilson
J E
JE
D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D001667:Binding, Competitive; D001921:Brain / Q000201:enzymology; D002478:Cells, Cultured; D004795:Enzyme Stability; D019298:Glucose-6-Phosphate / Q000031:analogs & derivatives; D005958:Glucosephosphates / Q000378:metabolism; D006593:Hexokinase / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D006600:Hexosephosphates / Q000494:pharmacology; D007527:Isoenzymes / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D010710:Phosphates / Q000378:metabolism; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013329:Structure-Activity Relationship
D005958:Glucosephosphates; D006600:Hexosephosphates; D007527:Isoenzymes; D010710:Phosphates; D011993:Recombinant Fusion Proteins; C000096:1,5-anhydroglucitol-6-phosphate; D019298:Glucose-6-Phosphate; D000255:Adenosine Triphosphate; C016473:glucose-1,6-bisphosphate; D006593:Hexokinase