Parameters Affecting the Restoration of Activity to Inactive Mutants of Thymidylate Synthase via Subunit Exchange:  Further Evidence That Thymidylate Synthase Is a Half-of-the-Sites Activity Enzyme

2-5 oligoadenylate (2-5 (A)) synthetases are major components of the antiviral pathways induced by interferons. In the presence of double-stranded RNA, they polymerize ATP to form 2-5 (A) oligomers that, in turn, activate the latent ribonuclease RNase L, causing mRNA degradation. These enzymes, unlike other nucleotidyl transferases, catalyze 2-5, not 3-5, phosphodiester bond formation between substrates bound to the acceptor and donor sites. Moreover, unlike other members of this extended family, the P69 isozyme of 2-5 (A) synthetase functions as a homodimer. Here, we report that the need for P69 dimerization is because of a crisscross enzyme reaction joining two substrate molecules bound to two opposite subunits. Consequently, although homodimers of mutants in the previously identified acceptor site, the donor site, or the catalytic site were inactive, selective heterodimers of the mutants were active because of subunit complementation. The catalytic site had to be present in the same subunit that contained the acceptor site, whereas the donor site had to be provided by the other subunit. These results allowed us to design a mutant protein that acted as a dominant-negative inhibitor of wt P69 but not of another isozyme of 2-5 (A) synthetase.

Section: Fig 3 Catalytic Efficiency Of Active Heterodimerssupporting

confidence: 64%

Crisscross Enzymatic Reaction between the Two Molecules in the Active Dimeric P69 Form of the 2′-5′ Oligodenylate Synthetase

Sarkar

Pal

Sen

2002

“…This proposal is based on structural evidence that suggests that TS binds ligands asymmetrically to each monomer's active site and mutagenesis studies in which an active site and non-active site TS dead mutant are combined to form a heterodimer with fully restored TS activity (17)(18)(19). To our knowledge, however, this study provides the first direct kinetic evidence for half-sites reactivity in TS enzymes.…”

Section: Discussionmentioning

confidence: 94%

“…It has been suggested that TS is a half-sites-reactive enzyme in which only one TS site of dimeric TS productively binds substrates (17)(18)(19). Until now, however, definitive kinetic evidence was lacking and the issue of whether TS is actually a half-sites-reactive enzyme has remained unresolved (9).…”

mentioning

confidence: 99%

Mechanistic Characterization of Toxoplasma gondiiThymidylate Synthase (TS-DHFR)-Dihydrofolate Reductase

Johnson

Hinz

Atreya

et al. 2002

Self Cite

This study describes the use of rapid transient kinetic methods to characterize the bifunctional thymidylate synthase-dihydrofolate reductase (TS-DHFR) enzyme from Toxoplasma gondii. In addition to elucidating the detailed kinetic scheme for this enzyme, this work provides the first direct kinetic evidence for the formation of a TS intermediate and for half-sites TS reactivity in human and Escherichia coli monofunctional TS and in T. gondii and Leishmania major bifunctional TS-DHFR. Comparison of the T. gondii TS-DHFR catalytic mechanism to that of the L. major enzyme reveals the mechanistic differences to be predominantly in DHFR activity. Specifically, TS ligand induced domain-domain communication involving DHFR activation is observed only in the L. major enzyme and, whereas both DHFR activities involve a rate-limiting conformational change, the change occurs at different positions along the kinetic pathway.

“…These results imply that the K282E/R283E TS-dead mutant is unable to form the covalent dUMP⅐CH 2 H 4 -folate⅐enzyme complex requisite properly for chemistry to take place. Restoration of Activity to Assess Global Stability-From studies in E. coli, it is known that TS, which exists as a dimer in most species, exhibits half-the-sites activity, meaning that at any given time, only one half of the TS dimer is kinetically competent (31,32). In E. coli TS it was shown that the amino acid Arg-126 participates in the catalytic site of the opposite half of the dimer and that a TS dimer in which both Arg-126 residues have been mutated to glutamic acid is TS-dead.…”

Section: Resultsmentioning

confidence: 99%

Probing Electrostatic Channeling in Protozoal Bifunctional Thymidylate Synthase-Dihydrofolate Reductase Using Site-directed Mutagenesis

Atreya

Johnson

Williamson

et al. 2003

In this study we used site-directed mutagenesis to test the hypothesis that substrate channeling in the bifunctional thymidylate synthase-dihydrofolate reductase enzyme from Leishmania major occurs via electrostatic interactions between the negatively charged dihydrofolate produced at thymidylate synthase and a series of lysine and arginine residues on the surface of the protein. Accordingly, 12 charge reversal or charge neutralization mutants were made, with up to 6 putative channel residues changed at once. The mutants were assessed for impaired channeling using two criteria: a lag in product formation at dihydrofolate reductase and an increase in dihydrofolate accumulation. Surprisingly, none of the mutations produced changes consistent with impaired channeling, so our findings do not support the electrostatic channeling hypothesis. Burst experiments confirmed that the mutants also did not interfere with intermediate formation at thymidylate synthase. One mutant, K282E/R283E, was found to be thymidylate synthase-dead because of an impaired ability to form the covalent enzyme-methylene tetrahydrofolate-deoxyuridate complex prerequisite for chemical catalysis.Electrostatic channeling is a mechanism proposed based on the crystal structure of bifunctional thymidylate synthase-dihydrofolate reductase (TS-DHFR) 1 from Leishmania major that would enable negatively charged dihydrofolate produced at the TS active site to be handed off along a series of solventexposed lysine and arginine residues to the DHFR active site, where it is converted to tetrahydrofolate (H 4 folate) (1).TS and DHFR are crucial enzymes, found in all species. TS represents the only means of de novo synthesis of 2Ј-deoxythymidylate (dTMP) for DNA synthesis, via reductive methylation of 2Ј-deoxyuridate (dUMP) with methylene tetrahydrofolate (CH 2 H 4 folate), producing dihydrofolate (H 2 folate) in the process. DHFR catalyzes the reduction of H 2 folate by NADPH to generate H 4 folate, used for one carbon unit transfer reactions in several biochemical processes, including thymidylate, purine, and amino acid biosynthesis. Only in protozoal parasites and some plants however, are TS and DHFR found on the same polypeptide chain, leading to the hypothesis that in these organisms, H 2 folate may be channeled from the TS active site to that of DHFR, never equilibrating with bulk solution (Fig. 1A). When the crystal structure of L. major TS-DHFR was solved (2.9 Å resolution), a 40 Å "electrostatic highway" across the surface of the protein was proposed as an explanation for how channeling may occur (1, 2). We sought to test the electrostatic channeling hypothesis with two parallel approaches. In this report we present results of mutagenesis of solvent-exposed basic residues comprising the electrostatic highway. In an earlier paper, we reported the findings from targeted molecular docking searches to identify small molecule inhibitors that bind in this region, as a means to block the putative channel (3).There are precedents for channeling among bifunction...