Isolation of a Covalent Steady-state Intermediate in Glutamate 60 Mutants of Thymidylate Synthase*

Glutamate 60 of thymidylate synthase coordinates a hydrogen bond network important in proton transfer reactions to and from the substrate dUMP. The E6OAand E60L mutants of Lactobacillus caaei thymidylate synthase catalyzed tritium exchange from [5-sHldUMP for solvent protons faster than dTMP formation, indicating accumulation of a steady-state intermediate and a change in partitioning of the intermediate. A covalent complex consisting of E60A or E60L thymidylate syn- thase, dUM.P, and the cofactor CH,H,folate was isolated on SDS-polyacrylamide gel electrophoresis and shown to be chemically and kinetically competent to form d m . These results provide proof of the formation of a covalent steady-state intermediate in the reaction pathway of thymidylate synthase and demonstrate that the rate-determining step in the mutants occurs during conversion of the covalent intermediate to dTMP. EC 2.1.1.45) the reduc-tive methylation of dUMP by CH,H,folate to give dTMP and H,folate. gained structure Sequences from most conserved of known enzymes x-ray crystal structures of several free compared with that of the wild-type TS-[6-3HlFdUMP-CH,H4folate covalent complex, which contains one FdUMP molecule/TS monomer. Coomassie-stained protein bands excised, solubilized Solv-able (DuPont NEN), and in 10 ml of Aquasol-2 (DuPont NEN). p~ E60A (0.5 p~ in covalent non-radioac- tive


Isolation of a Covalent Steady-state Intermediate in
Glutamate 60 Mutants of Thymidylate Synthase* (Received for publication, August 26, 1994) Weidong Huang and Daniel V. SantiS Thymidylate synthase (TS,' EC 2.1.1.45) catalyzes the reductive methylation of dUMP by CH,H,folate to give dTMP and H,folate. In recent years, much insight has been gained about the structure and mechanism of this enzyme. Sequences from over 20 sources have revealed that TS is among the most conserved of known enzymes (1,2), and x-ray crystal structures of several free and bound forms of the enzyme have pointed to key residues involved in substrate binding and catalysis (3-6). We have been particularly interested in correlations of structurefunction relationships of TS as probed by the consequences of mutagenesis. In the present work, we show that Glu-60 mutants of TS catalyze formation of an isolatable, covalent steadystate intermediate. This represents one of the few reports in which a mutation of an enzyme results in accumulation and isolation of a stable, normal steady-state intermediate (7, 8).
The currently accepted minimal mechanism of TS is depicted in Scheme I. After Michael addition of Cys-198, to C-6 of dUMP, * This work was supported by Public Health Service Grant CA-14394 from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.  The amino acid numbering used is that of L. casei TS.
CH,H,folate condenses with C-5 of dUMP to give the covalent ternary complex I11 as a steady-state intermediate; here, Cys-198 of the enzyme is covalently attached to C-6 of dUMP and the one-carbon unit of the cofactor to C-5 of dUMP. Intermediate I11 is directly analogous to the much studied ternary complex formed between TS, FdUMP, and CH,H,folate (9, 10). In the reaction pathway leading to dTMP, i t is proposed that the H-5 of intermediate I11 is removed as a proton, followed by p-elimination of H,folate and hydride transfer to give the products, dTMP and H,folate. As indicated, covalent bond changes are believed to be facilitated by water-mediated general acidbase-catalyzed proton transfers at 0 -4 a n d C-5 of the heterocycle (9). A complex containing TS, dUMP, and CH,H,folate, which is presumed to be 111, is isolatable by rapid acid quenching of ongoing TS reactions (11); however, the putative intermediate is formed and processed too rapidly to allow convenient study, and we were unable to isolate it on SDS-PAGE.
Crystallographic studies have shown that the completely conserved Glu-60 of TS is involved in an extensive hydrogen bond network that includes several conserved side chains of the enzyme, ordered water molecules, and the pyrimidine ring of the substrate ( Fig. 1) (5, 12). Mutation of Glu-60 leads to large losses in catalytic activity (13, 14), and it has been proposed that this residue plays a role in stabilizing the incipient negative charge at 0 -4 of dUMP (12) or aids in the opening of the imidazolidine ring of CH,H,folate (4). We show here that mutation of Glu-60 to Ala or Leu affects the partitioning of the putative steady-state intermediate I11 and thereby allows its isolation.
MATERIALS AND METHODS Mutagenesis and Protein Purification-The E60A and E60L mutants were constructed by cassette mutagenesis of the L. casei TS synthetic gene in plasmid pSCTS13 (15). The 56-base pair BcZIIPstI fragment of pSCTS13 was replaced by a "stuffer" oligonucleotide, to give pSCTS13-stuffer. The stuffer was obtained using a 58-nucleotide self-priming oligonucleotide that was filled using T4 DNA polymerase and digested by BcZI and PstI to generate cohesive ends (16). The StuY PstI component of the "stuffer" retains the wild-type TS sequence; the BcZUStuI component does not encode TS and has an unique NotI site for restriction purification (17). The unique StuI site was introduced into pSCTS13-stuffer to reduce the size of the synthetic oligonucleotide cassette needed for mutagenesis from 56 base pairs (BcZUPstI) in the original synthetic TS gene to 32 base pairs (BcZUStuI). A mutagenic oligonucleotide duplex cassette containing 5"GATCAAAAGCEG (or CTG)CTGCTGTGGTT-3' was inserted into the BcZI and StuI sites of pSCTSl3-stuffer; the bases underlined change codon 60 from Glu to Ala or Leu. The mutant enzymes were purified to homogeneity as described previously (18). Enzyme Assays-TS activity was monitored spectrophotometrically at 340 nm (19). The standard assay buffer for dTMP formation contained 50 m M TES, pH 7.4, 25 m M MgCl,, 6.5 m M formaldehyde, 1 m M EDTA, and 75 rn P-mercaptoethanol. TS activity of E60A or E60L mutants, which was too low to be measured spectrophotometrically, was monitored by HPLC analysis. HPLC was performed using a Rainin HPLC equipped with a Hewlett-Packard 1040A diode array detector (20). Isocratic separation of dUMP and dTMP was accomplished on an Alex Ultrasphere IP column using 5 m M KH,PO,, pH 7.0, 5 m M tetran-butylammonium sulfate, and 2.5% (vh) acetonitrile as the eluant with a flow rate of 1 mumin. Retention volumes for dUMP and dTMP TS, 400 p~ dUMP, and 560 p~ 16-3HlCH,H,folate (26.6 mCi/mmol) in standard TES assay buffer at 25 "C. Aliquots (15 pl) were denatured at various times and analyzed on 12% SDS-PAGE as described (20).
For quantitation and assessment of kinetic parameters of the E60A or E60L TS-[6-'H]dUMP-CH,H4folate covalent complex, the radioactivity associated with the protein band at its optimum formation was compared with that of the wild-type TS-[6-3HlFdUMP-CH,H4folate covalent complex, which contains one FdUMP molecule/TS monomer. Coomassie-stained protein bands were excised, solubilized using Solvable (DuPont NEN), and counted in 10 ml of Aquasol-2 (DuPont NEN). The reaction mixtures contained 4.5 p~ E60A or E60L TS, 200 PM [6-3HldUMP (0.5 CVmmol), and 400 p~ CH,H4folate in standard TES assay buffer at 25 "C. The rate constant for disappearance of the covalent complex was obtained by adding a 100-fold excess of non-radioactive dUMP after the maximum formation of the complex and then monitoring the first order rate decrease of the radioactivity associated with the complex. The apparent first order rate constant for formation of the covalent complex was calculated by dividing the initial rate of formation by the concentration of TS monomer.

RESULTS AND DISCUSSION
The TS reaction can be monitored by following either dTMP formation or the release of tritium from [5-3H]dUMP, which accompanies dTMP formation. With wild-type TS, the observed rates of these reactions are essentially identical (Table I), indicating that tritium release occurs concomitantly with methylation. With E60L and E60ATS, there was a retardation of both the CH,H,folate-dependent tritium release from [5-3HldUMP (600-1,400-fold) and dTMP formation (25,000-fold). Importantly, with the E60A and E60L mutants, the CH,H,folate-  dependent tritium release from [5-3HldUMP was 20-and 40fold faster, respectively, than dTMP formation. In the absence of cofactor, tritium release was negligible (~0.02 min"). The uncoupling of 5-tritium release from 5-methylation indicates a change in the partitioning of an intermediate in the reaction pathway.
The rapid release of tritium from [EI-~HI~UMP compared with dTMP formation by the Glu-60 mutants can only be explained by an exchange reaction, where the tritium of the substrate is replaced by solvent protons faster than by one-carbon units of the cofactor. Indeed, exchange of tritium from [5-3HldUMP was directly demonstrated by analysis of the HPLC-isolated substrate during the course of the reaction. From the reaction mechanism (Scheme I) it can be seen that the exchange likely occurs from (a) proton abstraction from I11 to give I V , followed by ( b ) re-protonation with water, and (c) reversal of intermediate I11 to yield dUMP. The tritium released from I11 must equilibrate with solvent protons in order to observe the exchange; if this were slow, the observed rate of tritium exchange would be a low estimate of the net rate of formation of 111. In itself, the cofactor-dependent exchange reaction provides evidence for the putative intermediate 111.
The observation that the E60A and E60L TS-catalyzed tritium exchange is faster than methylation indicates that the rate-determining step of dTMP formation is subsequent to formation of putative intermediate 111. This suggested to us that I11 may accumulate. A mixture containing E60L TS, [6-3H]dUMP, and CH,H,folate was incubated, denatured at various times, and subjected to SDS-PAGE. As shown in Fig.