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Primary structure and tetrahydropteroylglutamate binding site of rabbit liver cytosolic 5,10-methenyltetrahydrofolate synthetase.
No full textThe 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
Domain structure and function of 10-formyltetrahydrofolate dehydrogenase.
No full text10-Formyltetrahydrofolate dehydrogenase catalyzes the NADP(+)-dependent oxidation of 10-formyltetrahydrofolate to CO2 and tetrahydrofolate. Previous studies have shown that the enzyme binds the physiological pentaglutamate form of tetrahydrofolate product so tightly that it remains bound during size exclusion chromatography (Cook, R. J., and Wagner, C. (1982) Biochemistry 21, 4427-4434). In addition to the dehydrogenase activity, the enzyme from rat liver has been reported to exhibit both 10-formyltetrahydrofolate hydrolase and aldehyde dehydrogenase activities (Cook, R. J., Lloyd, R. S., and Wagner, C. (1991) J. Biol. Chem. 266, 4965-4973). We have purified the enzyme from rabbit liver and found that it catalyzes the same three reactions with similar kinetic constants and that it is a 99-kDa homotetramer, as reported previously for the rat and pig enzymes. Previous studies have suggested that the enzyme is composed of three domains and has separate folate binding sites for the dehydrogenase and hydrolase activities. We have investigated the domain structure of the rabbit enzyme. Differential scanning calorimetry reveals two thermal transitions, indicating the presence of two independently folded domains. The pentaglutamate form of tetrahydrofolate and NADP+ each stabilize one of the thermal transitions, showing that these ligands bind to separate domains. Limited proteolytic digestions by several proteases cleave the enzyme in a linker region between the two domains. After proteolytic cleavage, the domains no longer remain associated and do not catalyze the 10-formyltetrahydrofolate dehydrogenase reaction. Isolation and characterization of the intact domains revealed that the N-terminal domain only catalyzes the NADP(+)-independent 10-formyltetrahydrofolate hydrolase activity and the C-terminal domain only catalyzes the NADP(+)-dependent aldehyde dehydrogenase activity. The kinetic constants of these isolated domains are similar to those of the intact enzyme. Binding studies on the native enzyme using fluorescence and isothermal titration calorimetry indicated that the enzyme binds one molecule of tetrahydrofolate and two molecules of NADP+ per tetramer. Dissociation constants for both ligands were also determined by these methods
THERMODYNAMIC ANALYSIS OF THE BINDING OF THE POLYGLUTAMATE CHAIN OF 5-FORMYLTETRAHYDROPTEROYLPOLYGLUTAMATES TO SERINE HYDROXYMETHYLTRANSFERASE
No full textAn assay for homocysteine
No full textDetermination of homocysteine levels in cells and serum is important because high homocysteine is a risk factor for cardiovascular disease. The currently used methods for homocysteine analysis either are time consuming or rely on the use of expensive equipment. Described in this study is an enzymatic assay that determines levels of homocysteine in multiple samples in less than 30 min at levels from 5 to 50 pmol using only a spectrophotometer. The reproducibility of the assay is consistent with the other methods currently used. A second assay, that is about 5-fold more sensitive, follows the enzymatic catalyzed solvent exchange of protons on glycine, which requires a scintillation counter. Both the spectrophotometric and the radiometric methods are based on the conversion of 5-methyltetrahydrofolate to tetrahydrofolate by methionine synthase. The tetrahydrofolate is formed in stoichiometric amounts to the homocysteine in the sample. In the spectrophotometric method the tetrahydrofolate is used at catalytic levels by three enzymes to form a metabolic cycle that generates NADPH from NADP+. In the radiometric assay tetrahydrofolate is required for the enzymatic exchange of the pro 2S proton of glycine with solvent. L-Cysteine, at levels more than 30-fold higher than the upper level of homocysteine used in these assays, does not give any measurable response
Detection of isoAspartate Residues as Post-Translational Modification of Protein and Peptides
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5-formyltetrahydrofolate binding sites in different folate-dependent enzymes
No full text10-Formyltetrahydrofolate dehydrogenase has previously been identified as a tight binding protein of the polyglutamate forms of tetrahydrofolate (R. J. Cook and C. Wagner, Biochemistry 21, 4427-4434, 1982). Each subunit contains two independently folded domains connected by a linking peptide. By using the stable substrate and product analogs 10-formyl 5,8-dideazafolate and 5, 8-dideazafolate, respectively, we have determined that the tight binding folate site is separate from the catalytic site and that it is located on the N-terminal domain of the protein. This was achieved by cross-linking 10-formyl 5,8-dideazafolate to the dehydrogenase through the carboxyl group of the substrate analog. The cross-linked substrate analog was converted to the cross-linked product complex by adding either NADP+ or 2-mercaptoethanol, proving that the 10-formyl 5,8-dideazafolate was bound at the active site. With the active site cross-linked to 5,8-dideazafolate and not available for binding, the enzyme still bound 5, 8-dideazafolate-[3H]tetraglutamate tightly but noncovalently. Separation of the large and small domains by limited proteolysis showed that the tightly bound 5,8-dideazafolate-[3H]tetraglutamate was located on the small domain. The location of the cross-linked 10-formyl 5,8-dideazafolate at the active site was determined by amino acid sequencing of an isolated tryptic peptide
Serine hydroxymethyltransferase: role of the active site lysine in the mechanism of the enzyme.
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