Functional consequences of mutation of highly conserved serine residues, found at equivalent positions in the N- and C-terminal domains of mammalian hexokinases.
Despite the extensive sequence similarity between the N- and C-terminal halves of the 100-kDa molecular weight mammalian hexokinases (ATP:D-hexose 6-phosphotransferase, EC 2.7.1.1), reflecting their evolutionary origin by duplication and fusion of a gene coding for a smaller ancestral hexokinase, there is evidence for a functional division, with the C-terminal domain retaining a catalytic role while the N-terminal domain serves a regulatory function [binding of the product inhibitor, glucose 6-phosphate) (Glc-G-P)]. Conversion of Ser603 to Ala in the C-terminal domain of rat Type I hexokinase, expressed in COS-1 cells, resulted in drastic reduction of catalytic activity; Ser603 is analogous to Ser158, a residue of critical catalytic importance in the homologous yeast hexokinase. In contrast, conversion of Ser155 to Ala in the N-terminal domain (analogous to Ser603 in the C-terminal domain) of rat Type I hexokinase had no effect on catalytic activity or on inhibition of the enzyme by the Glc-6-P analog, 1,5-anhydroglucitol-6-P. Immunoreactivity with monoclonal antibodies recognizing conformationally sensitive epitopes was not affected, indicating that neither mutation resulted in gross structural perturbation. These results are consistent with the assignment of catalytic function, involving Ser603, to the C-terminal domain, and demonstrate that the analogous Ser155 is not critical for either catalytic or regulatory function. The Type I isozyme, expressed in COS-1 cells, retained the ability to bind to mitochondria in a Glc-6-P-sensitive manner, as previously found with the enzyme isolated from mammalian tissues.
Baijal
M
M
Department of Biochemistry, Michigan State University, East Lansing 48824.
Wilson
J E
JE
D000818:Animals; D001483:Base Sequence; D019298:Glucose-6-Phosphate; D005958:Glucosephosphates / Q000378:metabolism; D006593:Hexokinase / Q000737:chemistry* / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D008928:Mitochondria / Q000201:enzymology; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D011233:Precipitin Tests; D011994:Recombinant Proteins / Q000378:metabolism; D012694:Serine / Q000737:chemistry; D013329:Structure-Activity Relationship; D014162:Transfection
D005958:Glucosephosphates; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D012694:Serine; D019298:Glucose-6-Phosphate; D006593:Hexokinase