Primary structure and tetrahydropteroylglutamate binding site of rabbit liver cytosolic 5,10-methenyltetrahydrofolate synthetase.
The primary sequence of 5,10-methenyltetrahydrofolate synthetase from rabbit liver was determined by amino acid sequencing of the purified enzyme. The enzyme contains 201 amino acid residues with a predicted mass of 22,779 Da. The enzyme is located in the cytosolic fraction of liver homogenates. Carbodiimide-activated 5-formyltetrahydropteroylmonoglutamate and the pentaglutamate form of the substrate both irreversibly inactivate the enzyme by forming a covalent bond to Lys-18. Non-activated 5-formyltetrahydropteroylpentaglutamate protected against this inactivation. Substrate specificity studies showed that increasing the number of glutamate residues from zero to five on 5-formyltetrahydropteroate results in a 2 order of magnitude increase in the affinity of the substrate for the enzyme but only a 3-fold increase in the value of Vmax.
Dipartimento di Scienze Biochimiche, Università La Sapienza, Rome, Italy.
B
B
Maras
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P
Stover
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S
Valiante
D
D
Barra
V
V
Schirch
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D019731:Carbon-Nitrogen Ligases; D003488:Cyanogen Bromide; D003600:Cytosol / Q000201:enzymology; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D008025:Ligases / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D008099:Liver / Q000201:enzymology*; D013058:Mass Spectrometry; D008930:Mitochondria, Liver / Q000201:enzymology; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D011817:Rabbits; D013379:Substrate Specificity; D013384:Succinate Cytochrome c Oxidoreductase / Q000378:metabolism; D013763:Tetrahydrofolates / Q000378:metabolism*; D014357:Trypsin
D010446:Peptide Fragments; D013763:Tetrahydrofolates; C030371:5,6,7,8-tetrahydrofolic acid; D013384:Succinate Cytochrome c Oxidoreductase; D007770:L-Lactate Dehydrogenase; D014357:Trypsin; D008025:Ligases; D019731:Carbon-Nitrogen Ligases; C021557:5,10-methenyltetrahydrofolate synthetase; D003488:Cyanogen Bromide