The catalytic center of glucose-6-phosphatase. HIS176 is the nucleophile forming the phosphohistidine-enzyme intermediate during catalysis.
Glucose-6-phosphatase (G6Pase), a key enzyme in glucose homeostasis, is anchored to the endoplasmic reticulum by nine transmembrane helices. The amino acids comprising the catalytic center of G6Pase include Lys(76), Arg(83), His(119), Arg(170), and His(176). During catalysis, a His residue in G6Pase becomes phosphorylated generating an enzyme-phosphate intermediate. It was predicted that His(176) would be the amino acid that acts as a nucleophile forming a phosphohistidine-enzyme intermediate, and His(119) would be the amino acid that provides the proton needed to liberate the glucose moiety. However, the phosphate acceptor in G6Pase has eluded molecular characterization. To identify the His residue that covalently bound the phosphate moiety, we generated recombinant adenoviruses carrying G6Pase wild type and active site mutants. A 40-kDa [(32)P]phosphate-G6Pase intermediate was identified after incubating [(32)P]glucose 6-phosphate with microsomes expressing wild type but not with microsomes expressing either H119A or H176A mutant G6Pase. Human G6Pase contains five methionine residues at positions 1, 5, 121, 130, and 279. After cyanogen bromide cleavage, His(119) is predicted to be within a 116-amino acid peptide of 13.5 kDa with an isoelectric point of 5.3 (residues 6-121), and His(176) is predicted to be within a 149-amino acid peptide of 16.8 kDa with an isoelectric point of 9.3 (residues 131-279). We show that after digestion of a non-glycosylated [(32)P]phosphate-G6Pase intermediate by cyanogen bromide, the [(32)P]phosphate remains bound to a peptide of 17 kDa with an isoelectric point above 9, demonstrating that His(176) is the phosphate acceptor in G6Pase.
Ghosh
Abhijit
A
Section on Cellular Differentiation, Heritable Disorders Branch, NICHD, National Institutes of Health, Bethesda, Maryland 20892, USA.
Shieh
Jeng-Jer
JJ
Pan
Chi-Jiunn
CJ
Sun
Mao-Sen
MS
Chou
Janice Yang
JY
D000256:Adenoviridae / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001120:Arginine / Q000737:chemistry; D001665:Binding Sites; D015153:Blotting, Western; D019556:COS Cells; D002384:Catalysis; D020134:Catalytic Domain; D003488:Cyanogen Bromide / Q000378:metabolism / Q000494:pharmacology; D018076:DNA, Complementary / Q000378:metabolism; D005952:Glucose-6-Phosphatase / Q000737:chemistry* / Q000378:metabolism*; D019298:Glucose-6-Phosphate / Q000378:metabolism; D006639:Histidine / Q000031:analogs & derivatives* / Q000737:chemistry*; D006801:Humans; D006860:Hydrogen Bonding; D015151:Immunoblotting; D008239:Lysine / Q000737:chemistry; D008861:Microsomes / Q000378:metabolism; D008956:Models, Chemical; D008969:Molecular Sequence Data; D009154:Mutation
D018076:DNA, Complementary; D006639:Histidine; D019298:Glucose-6-Phosphate; D001120:Arginine; D005952:Glucose-6-Phosphatase; D008239:Lysine; D003488:Cyanogen Bromide; C005436:phosphohistidine