A stable binary complex between Leishmania major thymidylate synthase and the substrate deoxyuridylate. A slow-binding interaction.

E P Garvey,D V Santi

doi:10.1016/s0021-9258(18)48048-9

Abstract

The thymidylate synthase (TS) activity in Leishmania major resides on the bifunctional protein thymidylate synthase-dihydrofolate reductase (TS-DHFR). We have isolated, either by Sephadex G-25 chromatography or by nitrocellulose filter binding, a binary complex between the substrate deoxyuridylate (dUMP) and TS from L. major. The kinetics of binding support a "slow binding" mechanism in which dUMP initially binds to TS in a rapid, reversible pre-equilibrium step (Kd approximately 1 microM), followed by a slow first-order step (k = 3.5 X 10(-3) s-1) which results in the isolable complex; the rate constant for the dissociation of dUMP from this complex was 2.3 X 10(-4) s-1, and the overall dissociation constant was approximately 0.1 microM. The stoichiometry of dUMP to enzyme appears to be 1 mol of nucleotide bound/mol of dimeric TS-DHFR. Binary complexes between the stoichiometric inhibitor 5-fluorodeoxyuridylate (FdUMP) and TS, and between the product deoxythymidylate (dTMP) and TS were also isolated by nitrocellulose filter binding. Competition experiments indicated that each of these nucleotides were binding to the same site on the enzyme and that this site was the same as that occupied by the nucleotide in the FdUMP-cofactor X TS ternary complex. Thus, it appeared that the binary complexes were occupying the active site of TS. However, the preformed isolable dUMP X TS complex is neither on the catalytic path to dTMP nor did it inhibit TS activity, even though the dissociation of dUMP from this complex is several orders of magnitude slower than catalytic turnover (approximately 3 s-1). The results suggest that dUMP binds to one of the two subunits of the native protein in a catalytically incompetent form which does not inhibit activity of the other subunit.

Highlights

From the DeDartmnts of Biochemistrv and Bio-Dh”vsics and of Pharmaceutical Chemistry, University of California, San FrancisEo, Californh 94143
The bifunctional protein is a dimer, stoichiometry of dUMP to enzyme appears tobe 1mol consisting of identical subunits that each possess both activof nucleotide bound/mol of dimeric TS-DHFR
The preformedisolable dUMP-TS complex is neither on the catalytic path to dTMP nor did it inhibit TS activity, even though the dissociation of dUMP fromthis complex isseveral orders of magnitude slower than catalytic turnover(-3 s-l).The resultssuggest that dUMP binds toone of the two subunits of the native protein in a catalytically incompetent form which does not inhibit activityof the DHFR is similar to the higher order structures of the other synthases (8, 9)

Summary

A SLOW-BINDING INTERACTION*

From the DeDartmnts of Biochemistrv and Bio-Dh”vsics and of Pharmaceutical Chemistry, University of California, San FrancisEo, Californh 94143. Crude extract from wild-type cells was passed through DEAE-Sepharose and TS-DHFR eluted as previously described (6); thipsreparation was never in contact with exogenous folates Incubating this enzyme with [6-3H]dUMPresulted in formation of the binary complex. In experiments inwhich rates of dissociation were determined from a given complex, reaction mixtures were preincubated for 3 h, a length of time in which all complexes examined had fully formed, and a 500-fold excess of the unlabeled nucleotide was added to the reaction mixture; filter tide, and was shown to be protein-bound by nitrocellulosefilter binding These dataconfirmed that the isolation of the binary complex was notanartifact of nitrocellulose filter binding.

RESULTS

TSdUMP 2

Findings

DISCUSSION