THERMODYNAMIC ANALYSIS OF THE BINDING OF THE POLYGLUTAMATE CHAIN OF 5-FORMYLTETRAHYDROPTEROYLPOLYGLUTAMATES TO SERINE HYDROXYMETHYLTRANSFERASE

The thermodynamic parameters for the binding of 5-formyltetrahydrofola te (5-CHO-H(4)PteGlu(n)) and its polyglutamate forms to rabbit liver c ytosolic serine hydroxymethyltransferase (SHMT) were determined by a c ombination 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)P teGlu(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 i n the positive enthalpy with little change in entropy. A variety of an ions 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 bi nding 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), suggestin g that spacing of the negative charges may not be important in determi ning their effectiveness as inhibitors. Both the mono- and pentaglutam ate 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, Hi s, and Arg residues.