Spinach cytosolic fructose-1,6-bisphosphatase. Purification, enzyme properties and structural comparisons.
Cytosolic fructose-1,6-bisphosphatase from spinach leaves was purified to homogeneity and characterized. The pure enzyme has a subunit mass of 38 kDa, its Km values for fructose 1,6-bisphosphate and Mg2+ are 1.5 microM and 260 mM, respectively, and its Vmax is 110-120 units/mg. It is inhibited by fructose 2,6-bisphosphate and AMP with Ki values of 0.07 microM and 120 microM, respectively. About 90% of the primary structure of the spinach cytosolic fructose-1,6-bisphosphatase has been determined by amino-acid sequencing. The sequence data demonstrate that the cytosolic enzyme lacks the sequence insert characteristic of chloroplast fructose-1,6-bisphosphatase. The data include also the sequences of peptides containing all seven cysteine residues. Only two of the seven cysteines are conserved between the two isozymes, none of which is believed to be involved with the light regulation of the chloroplast enzyme. Sequence comparisons between the spinach cytosolic enzyme and gluconeogenic fructose-1,6-bisphosphatases from other species reveal similarity ranging over 47-54%, which is higher than the 40-45% similarity between the chloroplast enzyme and gluconeogenic fructose-1,6-bisphosphatases. However, similarity between these isozymes and Escherichia coli fructose-1,6-bisphosphatase are 44% and 47% for the cytosolic and chloroplast enzymes, respectively. Similarity between the cytosolic and chloroplast counterparts is 52%, indicating wide divergence between these two fructose-1,6-bisphosphatases.
Ladror
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US
Department of Biological Chemistry and Structure, University of Health Sciences, Chicago Medical School, IL 60064.
Latshaw
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SP
Marcus
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F
D000595:Amino Acid Sequence; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D003600:Cytosol / Q000201:enzymology; D006597:Fructose-Bisphosphatase / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000302:isolation & purification; D010447:Peptide Hydrolases; D010944:Plants / Q000201:enzymology*; D012689:Sequence Homology, Nucleic Acid
D010446:Peptide Fragments; D006597:Fructose-Bisphosphatase; D010447:Peptide Hydrolases