Thermodynamic analysis of the binding of the polyglutamate chain of 5-formyltetrahydropteroylpolyglutamates to serine hydroxymethyltransferase

The thermodynamic parameters for the binding of 5-formyltetrahydrofolate (5-CHO-H(4)PteGlu(n)) 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-H(4)PteGlu(n) to SHMT exhibits both positive enthalpy and entropy, showing that binding is entropically driven. 5-CHO-H(4)PteGlu(5) has a 300-fold increased affinity for SHMT compared to 5-CHO-H(4)PteGlu. 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-H(4)PteGlu(5) with K(i) values in the 10-20 mM range. Anions are ineffective inhibitors of 5-CHO-H(4)PteGlu binding to SHMT, showing that anions compete for the polyglutamate binding site. There was little difference in the K(i) values for a series of dicarboxylic acids as inhibitors of 5-CHO-H(4)PteGlu(5), 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-H(4)PteGlu(n) were cross-linked to SHMT by a carbodiimide reaction to Lys-450 which resides in a stretch of Lys, His, and Arg residues.