Mode of binding of folate analogs to thymidylate synthase. Evidence for two asymmetric but interactive substrate binding sites.

I.K Dev,W.S Dallas,R Ferone,M Hanlon,D.D Mckee,B.B Yates

doi:10.1016/s0021-9258(17)42108-9

Abstract

Human thymidylate synthase is a polymeric protein composed of two subunits with identical primary structures. In this study we determined the binding affinities of 5,10-methylene tetrahydropteroyltetraglutamate (folate substrate) and a group of close structural folate analog inhibitors. Thymidylate synthase bound both mono and polyglutamylated folate substrates and analogs more tightly in the presence of deoxyuridylate. These results and product inhibition studies confirmed that the orders of substrate addition and product release from thymidylate synthase were similar for mono and polyglutamylated substrates. Equilibrium dialysis studies showed that the folate substrate in a ternary complex with deoxyuridylate bound to one of the subunits (site A) with a Kd of 720 nM. The binding of the substrate to the second subunit (site B) was much weaker, and the Kd could not be determined by this method. However, dissociation constants for each subunit could be measured for the folate analog inhibitors, and, depending on the inhibitor, the relative Kd value for each subunit varied substantially. For example, formyl-5,8-dideazafolate and tetraglutamylated 10-propargyl-5,8-dideazafolate bound to both sites with similar Kd values, whereas D1694Glu4 bound to subunit A with a higher affinity (Kd = 1.0 nM) than to subunit B (Kd = 30 nM). In contrast, 1843U89 (mono or diglutamylated form) had a much higher affinity for subunit B (Kd approximately 0.1 nM) compared with subunit A (Kd approximately 400 nM). Enzyme inhibition kinetic analyses showed that the Ki values of 1843U89 were quite low (0.1 nM) and that the inhibition was noncompetitive. In contrast, the other folate analogs inhibited the enzyme via mixed inhibition (i.e. both the Km for the folate substrate and the Vmax were altered). We conclude that the two subunits of thymidylate synthase bind folate substrates and analogs differently and that the asymmetric binding of the ligands is the major factor that determines the inhibition kinetics of each folate analog inhibitor.

Highlights

Human thymidylate synthase is a polymeric protein ductive methylation of deoxyuridylate to thymidylate composed of two subunits with identical primary struc- whileconverting the cofactor 5,lO-methylene-H4PteGlu t o tures
Steady-state kinetic parameters for the recombinant human TS were K, = 2.5 p ~K, ((6R)5,10-methylene-H4PteGlulf= 12 p ~ T.he specific activity of different preparations varied between0.8 and 1.2 pmol/min/mg of the recombinant human TS at 3"7C.The kinetic parameters mg/mlbovine serum albumin, 1,400 nM 6-[3HldUMP (15 Ci/mmol, Moravek Biochemicals), 30n~ human TS, and various amounts of the folate analogs a t 25 "C.The rate of dissociation of the binary complex of human TS.[3HldUMPor dUMP froma ternary complex wasstudied by the addition of a 1,000-fold excessof unlabeled dUMP tothe preformed complexes
The results obtained from the measurement of binding of folate substrates and analogs to dimeric TS suggest that the two substrate binding sites exhibit ligand-induced negativecooperativity

Summary

Introduction

Human thymidylate synthase is a polymeric protein ductive methylation of deoxyuridylate (dUMP) to thymidylate composed of two subunits with identical primary struc- whileconverting the cofactor 5,lO-methylene-H4PteGlu t o tures. The binding of [14ClD1694Glu4a, close structural analog of the 1843U89 ( K d = 0.1 m,gel permeation) or PDDF ( K d = 46 m, folate cofactor,to human TS (40 nM) in a ternary complex with equilibrium dialysis)with a stoichiometry of 0.78 and 1.2mol of dUMP was determined by equilibrium dialysis, with inhibitor inhibitor per mol ofdimeric enzyme,respectively (Table11).The concentrations ranging upto 400 nM.

Results

Conclusion