title: Thermodynamic analysis of the binding of the polyglutamate chain of 5-formyltetrahydropteroylpolyglutamates to serine hydroxymethyltransferase.
abstract: The thermodynamic parameters for the binding of 5-formyltetrahydrofolate (5-CHO-H4PteGlun) and its polyglutamate forms to rabbit liver cytosolic serine hydroxymethyltransferase (SHMT) were determined by a combination of isothermal titration calorimetry and spectrophotometry. Binding of 5-CHO-H4PteGlun to SHMT exhibits both positive enthalpy and entropy, showing that binding is entropically driven. 5-CHO-H4PteGlu5 has a 300-fold increased affinity for SHMT compared to 5-CHO-H4PteGlu. This increase in affinity is due primarily to a decrease in the positive enthalpy with little change in entropy. A variety of anions inhibit the binding of 5-CHO-H4PteGlu5 with Ki values in the 10-20 mM range. Anions are ineffective inhibitors of 5-CHO-H4PteGlu binding to SHMT, showing that anions compete for the polyglutamate binding site. There was little difference in the Ki values for a series of dicarboxylic acids as inhibitors of 5-CHO-H4PteGlu5, suggesting that spacing of the negative charges may not be important in determining their effectiveness as inhibitors. Both the mono- and pentaglutamate derivatives of 5-CHO-H4PteGlun were cross-linked to SHMT by a carbodiimide reaction to Lys-450 which resides in a stretch of Lys, His, and Arg residues.
authors.affiliation: Institute for Structural Biology and Drug Discovery, Virginia Commonwealth University, Richmond 23298-0614, USA.
authors.forename: T
authors.initials: T
authors.lastname: Huang
authors.forename: C
authors.initials: C
authors.lastname: Wang
authors.forename: B
authors.initials: B
authors.lastname: Maras
authors.forename: D
authors.initials: D
authors.lastname: Barra
authors.forename: V
authors.initials: V
authors.lastname: Schirch
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002151:Calorimetry; D005575:Formyltetrahydrofolates / Q000737:chemistry*; D012696:Glycine Hydroxymethyltransferase / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D008099:Liver / Q000201:enzymology; D008969:Molecular Sequence Data; D009994:Osmolar Concentration; D011099:Polyglutamic Acid / Q000737:chemistry* / Q000378:metabolism; D011817:Rabbits; D013056:Spectrophotometry, Ultraviolet; D013763:Tetrahydrofolates / Q000737:chemistry; D013816:Thermodynamics
chemical_list: D005575:Formyltetrahydrofolates; D013763:Tetrahydrofolates; D011099:Polyglutamic Acid; D012696:Glycine Hydroxymethyltransferase; C005984:5-methyltetrahydrofolate