Isolation of a human liver fructose-1,6-bisphosphatase cDNA and expression of the protein in Escherichia coli. Role of ASP-118 and ASP-121 in catalysis.
A cDNA encoding human liver fructose-1,6-bisphosphatase was isolated from a lambda gt11 library by screening with a rat liver fructose-1,6-bisphosphatase cDNA. The cDNA (1421 base pairs) contains an open reading frame encoding 337 amino acids, corresponding to a protein with an estimated molecular weight of 36,697. Its primary sequence is highly homologous to that of the pig kidney and rat liver enzymes. The human liver cDNA was used to construct a T7 RNA polymerase-transcribed expression vector, and the enzyme was expressed in Escherichia coli BL21 (DE3). Approximately 50% of the expressed human fructose-1,6-bisphosphatase was soluble and enzymatically active, and the enzyme was purified to homogeneity by heat treatment, ammonium sulfate fractionation, and substrate/AMP elution from carboxymethyl-Sephadex. Expressed human liver fructose-1,6-bisphosphatase had a specific activity (9.8 mumol/min/mg of protein) that was half that of the rat liver enzyme, but had an identical Km for substrate. However, the human enzyme was more sensitive to inhibition by fructose-2,6-bisphosphate (Ki = 0.3 microM) and AMP (Ki = 12 microM) than the rat liver form (fructose 2,6-P2, Ki = 4 microM; AMP, Ki = 40 microM). Crystallographic analyses have suggested that Asp-118 and Asp-121 are catalytic residues located in a negatively charged pocket that binds divalent metal cations. These residues were mutated to alanine, and the E. coli-expressed mutant enzymes were purified to homogeneity. The Asp-118-->Ala and Asp-121-->Ala mutants had 1/5000 and 1/20,000 lower Kcat values than the wild-type enzyme, respectively, consistent with their critical role in fructose-1,6-bisphosphatase catalysis.
Department of Physiology and Biophysics, State University of New York, Stony Brook 11794.
M R
MR
el-Maghrabi
M
M
Gidh-Jain
L R
LR
Austin
S J
SJ
Pilkis
D000595:Amino Acid Sequence; D000818:Animals; D001224:Aspartic Acid; D001483:Base Sequence; D002645:Chickens; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics*; D004591:Electrophoresis, Polyacrylamide Gel; D004790:Enzyme Induction; D006597:Fructose-Bisphosphatase / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D006801:Humans; D007668:Kidney / Q000201:enzymology; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D017386:Sequence Homology, Amino Acid; D013552:Swine
D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D001224:Aspartic Acid; D004247:DNA; D006597:Fructose-Bisphosphatase