Functional Effects of Amino Acid Substitutions at Residue 33 of Human Thymidylate Synthase

ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS

Vol. 342, No. 2, June 15, pp. 338–343, 1997 Article No. BB970116

Functional Effects of Amino Acid Substitutions at Residue 33 of Human Thymidylate Synthase1 R. Todd Reilly,2 Antonia M. Forsthoefel, and Franklin G. Berger3 Department of Biological Sciences, University of South Carolina, Columbia, South Carolina 29208

Received February 11, 1997, and in revised form March 26, 1997

Fluorinated pyrimidines, such as 5-fluorouracil (FUra) and 5-fluoro-2*-deoxyuridine (FdUrd), are cytotoxic to cells as a consequence of generation of 5-fluoro-2*-deoxyuridylate (FdUMP), which is a mechanism-based inhibitor of the enzyme thymidylate synthase (TS). FdUMP inhibits TS via its binding into a stable inhibitory ternary complex (ITC) with the enzyme and the cosubstrate N5,N10-methylene-5,6,7,8-tetrahydrofolate (CH2H4PteGlu). In previous studies, we identified a naturally occurring mutant form of human TS that contains a Tyr r His substitution at residue 33 and confers relative resistance to FdUrd in both mammalian and bacterial cells. Kinetic studies indicated that the equilibrium dissociation constant (Kd) for binding of FdUMP into the ITC is altered in the mutant enzyme. In the current investigation, we have examined the kinetics of FdUMP binding into covalent binary complexes, i.e., in the absence of CH2H4PteGlu. Our results showed that although the rate constants for binary FdUMP binding (i.e., kon and koff) are altered by the Tyr r His substitution, there is no measurable effect on the overall Kd . Analysis of a number of other amino acid substitutions at residue 33 indicated that maximal enzyme accumulation and function requires a bulky, hydrophobic side chain at this site. q 1997 Academic Press Key Words: thymidylate synthase; fluoropyrimidines; 5-fluoro-2*-deoxyuridylic acid; N5,N10-methylene-5,6,7,8tetrahydrofolic acid.

Fluoropyrimidine antimetabolites, such as FUra4 and FdUrd, have been useful in the clinical management 1 This work was supported by a grant from the National Institutes of Health (CA44013). 2 Present address: Ross Building, Room 350, Johns Hopkins University, 720 Rutland Avenue, Room 350, Baltimore, MD 21205. 3 To whom correspondence should be addressed. Fax: 803-7774002. E-mail: [email protected]. 4 Abbreviations used: TS, thymidylate synthase; FUra, 5-fluorouracil; FdUrd, 5-fluoro-2*-deoxyuridine; FdUMP, 5-fluoro-2*-deoxyuridylic acid; CH2H4PteGlu, N5N10-methylene-5,6,7,8-tetrahydrofolic acid; ITC, inhibitiory ternary complex; kon , rate constant for ligand

of cancer, particularly of the breast and colon (1, 2). These agents are cytotoxic to growing cells as a consequence of their ability to form FdUMP, a mechanismbased inhibitor of the enzyme thymidylate synthase (TS; EC 2.1.1.45). TS catalyzes the synthesis of dTMP via the reductive methylation of dUMP by CH2H4PteGlu (3, 4). The enzyme, which exists as a homodimer, is indispensable in the de novo synthesis of dTMP and therefore plays an important role in DNA replication in actively dividing cells. FdUMP-mediated inhibition of TS occurs through formation of a covalent ternary complex with the enzyme and CH2H4PteGlu (1–4). This complex, termed the ITC, is held together by two covalent bonds: one between the C6 of FdUMP and a conserved cysteine sulfhydryl in the enzyme and the other between the C5 of the nucleotide and the methylene carbon of CH2H4PteGlu (1–4). The high stability of the ITC essentially titrates out available active sites, leading to a nearly complete blockage of dTMP synthesis. Recent stopped-flow chemical quench experiments (5) have indicated that covalent binding of FdUMP to the TS dimer occurs in a biphasic manner. An initial, rapid phase represents ITC formation at the first subunit of the dimer; this is followed by a second, slower phase representing ITC formation at the second subunit (5). The maximum stoichiometry of covalent FdUMP binding is 1.7/dimer (5–7). The biphasic nature of ITC formation indicates that the two TS subunits may be nonequivalent, a feature that has been noted in previous studies conducted by a number of laboratories (3, 8–14). Since crystal structure studies have shown that the two subunits of the ligand-free enzyme are identical in conformation (3, 15–19), the nonequivalence with regard to ITC formation may reflect cooperative interactions between the two subunits, where ligand binding at the first subunit alters the nature of ligand binding at the second (5). association; koff , rate constant for ligand dissociation; Kd , equilibrium dissociation constant; SDS–PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; TCA, trichloroacetic acid.

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Earlier, we identified a naturally occurring mutant form of TS that contains a Tyr r His substitution at residue 33 and confers relative resistance to FdUrd in both mammalian and bacterial cells (20–23). Tyr33 is a highly conserved residue that is not in the active site proper, and plays no direct role in ligand binding or catalysis (3, 20–22). FdUrd resistance in cells expressing the His33 enzyme, which has a lower kcat relative to the wild-type Tyr33 form, derives from alterations in the kinetics of FdUMP binding (5, 23). Rapid chemical quench studies (5) are consistent with the notion that the effects of the His33 substitution on ITC formation reflect alterations in the degree of ligand-induced negative cooperativity that occurs between the two TS subunits. To date, functional comparisons of the Tyr33 and His33 enzymes have focused on ternary complexes containing the enzyme, FdUMP, and CH2H4PteGlu (5, 20– 23). It has been known for quite some time that FdUMP is capable of forming a covalent binary complex with TS in the absence of CH2H4PteGlu (24–27). Presently, we report that the Kd for FdUMP binding into a binary complex is essentially unaffected by the Tyr33 r His substitution, indicating that the impact of this amino acid change on overall FdUMP affinity requires CH2H4PteGlu. In addition, we have examined the properties of a number of mutant TS molecules containing amino acid substitutions at residue 33. Our analysis shows that maximal enzyme function requires a bulky hydrophobic side chain at this site. The implications of these results to TS function, particularly the effects of the His33 substitution, are discussed. MATERIALS AND METHODS The human TS-expressing plasmid pDHTS-S1 was maintained in Escherichia coli strain x2913 (28). Overnight cultures (2.5 ml) were inoculated into 250 ml of L-broth containing 100 mg/ml ampicillin; after 24–26 h, cells were harvested by centrifugation at 3000g for 10 min, washed once with phosphate-buffered saline, and resuspended in 15 ml of 50 mM Tris–OH (pH 7.4) containing 1 mM Na2 EDTA, 0.1 mM phenylmethylsufonyl fluoride, 0.2% 2-mercaptoethanol, and 10 mM dithiothreitol. The cells were sonicated on ice using a Branson model 450 sonifier (4 cycles, 30 s/cycle, power level 6; duty cycle, 60%). Cellular debris was removed by centrifugation at 10,000g for 30 min, and protein concentrations in the resulting extracts were determined using the Bradford method (29). Glycerol was added to a final concentration of 5%. Extracts prepared in this manner could be stored at 0707C for at least 3 months without significant loss of activity. TS enzyme concentrations in the extracts were determined by the FdUMP binding assay (30), as described previously (20). Reaction mixtures (150 ml) contained Morrison buffer (120 mM Tris, 60 mM Mes, 60 mM acetic acid, pH 7.2), 15–20 mM [6-3H]FdUMP (16.6 Ci/ mmol), 200 mM CH2H4PteGlu, 500 mg/ml bovine serum albumin, and 200–400 mg extract protein. After incubation for 30 min at 257C, enzyme-bound radioactivity was precipitated by addition of 37.5 ml 50% trichloroacetic acid (TCA), and scintillation counting of the washed pellet. The concentration of TS dimers was calculated assuming a maximal FdUMP binding stoichiometry of 1.7/dimer (5–7). TS activity levels were measured by the tritium release assay (31), as described previously (32). Reaction mixtures (300 ml) contained

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Morrison buffer (pH 7.2), 50 mM [5-3H]dUMP (16.6 Ci/mmol), 300 mM CH2H4PteGlu, 500 mg/ml bovine serum albumin, and 115–130 mg extract protein. Mixtures were incubated at 377C, and [3H]H2O water was measured by charcoal extraction (31, 32). One unit of activity is an amount of enzyme that catalyzes production of 1 mmol of [3H]H2O per minute. kcat values for TS in crude extracts were calculated from the ratios of enzyme activity (as determined from the tritium release assay) to concentration of enzyme dimers (as determined from the FdUMP binding assay); values were expressed in units of sec01. Purification of the Tyr33 and His33 forms of human TS was accomplished by a two-step protocol involving Cibacron blue–Sepharose chromatography followed by methotrexate–Sepharose affinity chromatography. Details of the purification have been described previously (5). Purity of enzyme preparations was assessed by SDS– PAGE. Scatchard analyses were carried out as previously described (5), except that CH2H4PteGlu was omitted from the reaction mixtures. Concentrations of [6-3H]FdUMP ranged between 5 and 50 nM. The binding ratio, Y, which is the number of bound FdUMP molecules per enzyme dimer, was calculated and plotted vs Y/[free FdUMP] (5). The rate constant kon , describing FdUMP association into a covalent binary complex with TS, was determined in 500-ml reaction mixtures containing Morrison buffer (pH 7.2), 25 mM [6-3H]FdUMP (16.6 Ci/mmol), and 25 nM TS. Mixtures were incubated at 77C for various times, and the amount of enzyme-bound radioactivity was measured by addition of 125 ml 50% TCA and scintillation counting of the washed pellet. kon values were calculated from the following equation (5): [FdUMP0]([E0] 0 [X]) 1 ln Å kont [E0] 0 [FdUMP0] [E0]([FdUMP0] 0 [X])

[1]

where [E0] is the initial concentration of enzyme binding sites, [FdUMP0] is the initial FdUMP concentration, and [X] is the concentration of binary complex at time t (5). The slope of a straight-line plot of Eq. [1] provides an estimate of kon . koff , the rate constant for FdUMP dissociation from the binary complex, was measured by FdUMP exchange. Covalent binary complexes were generated in 500-ml reaction mixtures containing Morrison buffer (pH 7.2), 25 mM [6-3H]FdUMP, and 25 nM TS. After incubation at 77C for 1.5 h, unlabeled FdUMP was added to a final concentration of 2.5 mM; at various times, 100-ml aliquots were removed and added to 25 ml 50% TCA to precipitate binary complexes. The amount of radioactivity remaining in the complexes was determined by scintillation counting of the isolated, washed pellet; koff values were determined by plotting ln [bound FdUMP] as a function of time. Mutations were introduced into the TS polypeptide by oligonucleotide-directed mutagenesis of plasmid pDHTS-S1 (28), using a kit from Amersham, Inc., as described previously (22). Mutant plasmids were identified by colony hybridization using the mutant oligonucleotide as probe and verified by DNA sequencing. They were tested for their ability to complement a thyA0 mutation in E. coli by transformation into the thymine-requiring strain x2913 (28). Transformants were plated onto L-agar containing 100 mg/ml ampicillin, and individual colonies were tested for thymine-independent growth on minimal agar plates containing 100 mg/ml ampicillin. For Western blotting, equal amounts of extract protein (10 mg) were denatured and resolved in a 12.5% SDS–PAGE gel. Proteins in the gel were electrophoretically transferred to a nitrocellulose filter in transfer buffer (25 mM Tris–Cl, pH 8.5, 192 mM glycine, 20% methanol) for 30 min at 15 V using a Bio-Rad Trans-Blot SD apparatus. The filter was incubated for 1 h at room temperature in blocking solution (phosphate-buffered saline containing 5% nonfat dry milk and 0.1% Tween 20) and washed four times in phosphatebuffered saline/0.1% Tween 20. The filter was incubated overnight at room temperature in 10 ml of culture supernatant from hybridoma

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FIG. 1. Scatchard analysis of FdUMP binding into a binary complex with TS. Binding of [3H]FdUMP to the purified Tyr33 (s) and His33 (m) forms of TS was measured as described under Materials and Methods. Y indicates the binding ratio, i.e., the number of bound FdUMP molecules per enzyme dimer (10).

cell line D3B31, which secretes a monoclonal antibody against human TS; the cell line was provided by Dr. S. Berger. Following the incubation, the blot was washed as above and incubated for 1 h at room temperature in a solution containing horseradish peroxidaselinked goat anti-mouse IgG (Bio-Rad; 1:1000 dilution in phosphatebuffered saline/0.1% Tween 20). After four washes in phosphatebuffered saline/0.1% Tween 20, the TS polypeptide was visualized by staining in 4-chloro-1-naphthol for 2–5 min.

RESULTS

FdUMP Binding into a Binary Complex with TS Purified preparations of the Tyr33 and His33 enzymes were used to determine the Kd values for covalent binary complex formation between FdUMP and TS. Enzymes were incubated in various concentrations of [6-3H]FdUMP, and formation of covalent binary complexes was measured by TCA precipitation. The Scatchard plot shown in Fig. 1 allowed estimating Kd values of 0.48 { 0.05 mM for the Tyr33 enzyme and 0.49 { 0.05 mM for the His33 enzyme. Maximal binding stoichiometry was 0.55/dimer for each enzyme. Thus, the Kd for FdUMP binding into a binary complex with TS appears to be identical for both enzymes. As an alternative method for determining Kd , we measured kon (the rate constant for FdUMP binding into a binary complex with TS) and koff (the rate constant for FdUMP dissociation from the binary complex); Kd was calculated from the ratio of these rate constants (i.e., Kd Å koff/kon). To measure kon , purified enzymes were incubated for various times at 77C in the presence of excess [6-3H]FdUMP, and covalent complex formation was measured by TCA precipitation (see Materials and Methods). From the slopes of the plot in Fig. 2, kon was estimated to be 0.194 { 0.005 mM01 min01 for the Tyr33 form and 0.074 { 0.007 mM01 min01 for the His33 form. koff was determined from rates of

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FIG. 2. Determination of kon for FdUMP binding into a binary complex with TS. Rates of [3H]FdUMP binding to purified preparations of the Tyr33 (s) and His33 (m) forms of TS were measured as described under Materials and Methods. The value Z represents the left side of Eq. [1] (see Materials and Methods).

exchange of enzyme-bound FdUMP in preformed binary complexes. Complexes labeled with [6-3H]FdUMP as described under Materials and Methods were incubated in a 1000-fold excess of unlabeled FdUMP, and the concentration of enzyme-bound radioactivity was measured by TCA precipitation. The koff values, calculated from the plot shown in Fig. 3, were 0.092 { 0.004 min01 for the Tyr33 enzyme and 0.035 { 0.003 min01 for the His33 form, indicating a 2.6-fold slower dissociation rate from the mutant enzyme. Kd values, as determined from the koff/kon ratios, were 0.47 and 0.48 mM for the Tyr33 and His33 forms, respectively. Thus, although the Tyr33 r His substitution results in changes in both kon and koff , there is no net change in Kd .

FIG. 3. Determination of koff for FdUMP dissociation from the binary complex. Binary complexes containing [3H]FdUMP bound to either the Tyr33 (s) or the His33 (m) form of TS were prepared; rates of exchange by unlabeled nucleotide were measured as described under Materials and Methods.

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Properties of Mutant TS Molecules Containing Amino Acid Substitutions at Residue 33

Enzyme

Thymine independencea

TS levelb

kcatc

Tyr33 Phe33 His33 Trp33 Leu33 Ile33 Val33 Ala33 Ser33 Asp33 Asn33 Lys33 Gly33

/ / / / / / / 0 0 0 0 0 0

417 630d 85 132 157 52 38 õ1 õ1 õ1 õ1 õ1 õ1

1.59 { 0.18 1.5d 0.12 { 0.01 0.46 { 0.05 0.65 { 0.06 0.69 { 0.03 0.38 { 0.01 —e —e —e —e —e —e

a The ability to grow in the absence of exogenous thymine is indicated by (/); the lack of such ability is indicated by (0). b Values represent pmol TS dimer/mg total extract protein, as determined by the FdUMP binding assay (see Materials and Methods); under the conditions utilized, the minimum level of detection is about 1 pmol/mg. c Values are in units of s01 ({standard error). d Data taken from Reilly et al. (5). e Not measurable due to the lack of detectable enzyme.

Complementation of Thymine Auxotrophy by TS Molecules Containing a Variety of Amino Acid Subsitutions at Residue 33 Residue 33 of human TS is nearly invariant among the approximately thirty TS molecules that have been sequenced. The only exception is the enzyme from Lactococcus lactis, which has phenylalanine rather than tyrosine at this site (3). Human TS with a Tyr33 r Phe mutation exhibits properties that are essentially indistinguishable from the wild-type enzyme (5, 23). In light of the functional changes elicited by the His33 replacement, it was of interest to assess the impact of other amino acid substitutions at residue 33 within the TS polypeptide. Ten mutant plasmids encoding TS molecules with different amino acid substitutions at residue 33 were produced, and each was tested for its ability to complement Escherichia coli x2913 cells, which are TS-deficient due to a thyA0 mutation, and require thymine for growth (28). Earlier studies had shown that the wildtype Tyr33 enzyme, as well as the mutant His33 and Phe33 forms, are fully capable of supporting growth of x2913 cells in thymine-free medium (22). Four additional mutant TS molecules (Leu33, Ile33, Val33, and Trp33) were likewise capable of supporting thymine-independent growth (Table I). Of interest is the fact that the side chains of these mutants contain bulky hydrophobic amino acids. Six other mutants did not support growth in the absence of exogenous thymine. The side chains of these mutants

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were polar (Ser33, Asn33), charged (Asp33, Lys33), or small (Ala33, Gly33). The results, in total, indicate that maintenance of a functional TS molecule requires a bulky, hydrophobic side chain at residue 33. Characterization of Mutant TS Expression All of the mutants that complemented growth of thyA0 bacteria contained measurable TS activity, as determined by FdUMP binding and tritium release assays in crude cell extracts (Table I). TS concentrations in the mutants ranged from about 0.3 to 1.0% of total protein, levels which are lower than that for the Tyr33 enzyme. The mutants that failed to support thymine-independent growth contained no detectable activity (Table I). Earlier it was shown that the catalytic efficiency (kcat) for the His33 form of TS is reduced by about 10- to 12fold relative to that for the Tyr33 and Phe33 enzymes (5, 23). For each of the mutants that retained TS function, we calculated kcat on the basis of the ratio of catalytic activity to FdUMP binding activity in crude extracts (see Materials and Methods); this normalizes for variation in TS concentrations among the various extracts. kcat values determined with crude extracts are very similar to those determined with purified enzymes (22, 23),5 so that use of crude preparations is a valid means of assessing catalytic efficiencies. As shown in Table I, the His33, Leu33, Ile33, Val33, and Trp33 mutants had kcat values that were 2- to 13-fold lower than that for the wild-type or Phe33 enzyme. Thus, though these mutant TS molecules are functional, they catalyze the TS reaction with reduced efficiencies. Western blotting using a human TS-specific monoclonal antibody as probe was conducted to determine the size and concentration of the TS molecule in extracts of bacteria harboring the various mutant plasmids. In all extracts, the antibody recognized a 36-kDa polypeptide (Fig. 4), which is the size expected for human TS (5, 23). However, the concentrations of the mutant TS enzymes were low compared to the Tyr33 form (Fig. 4); in particular, little or no enzyme was detected in extracts of mutants that failed to complement thyA0 E. coli (Fig. 4, bottom). Similar results were obtained using a polyclonal antibody (data not shown). Thus, the absence of detectable TS activity in extracts of the noncomplementing mutants (Table I) is due in large part to exceedingly low enzyme concentrations. The fact that the enzyme levels correlate with the chemical nature of the amino acid side chain at residue 33 makes it likely that the low TS concentrations reflect alterations in enzyme structure, rather than enzyme synthesis.

5

A. Forsthoefel, unpublished results.

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FIG. 4. Western blot analysis of TS levels. Samples were subjected to SDS–PAGE, transferred to nitrocellulose filters, and analyzed for the TS polypeptide by Western blotting using a human TS-specific monoclonal antibody as probe (see Materials and Methods for details). (Top) Purified human TS (lane 1): cell-free extracts of E. coli expressing the Tyr33 (lane 2), Trp33 (lane 3), Leu33 (lane 4), Ile33 (lane 5), and Val33 (lane 6) enzymes. (Bottom) Purified human TS (lane 1): cell-free extracts of E. coli expressing the Tyr33 (lane 2), Asp33 (lane 3), Asn33 (lane 4), Gly33 (lane 5), Lys33 (lane 6), Ala33 (lane 7), and Ser33 (lane 8) enzymes. Molecular weight markers are indicated to the left.

DISCUSSION

Previous analyses had indicated that the Tyr33 r His substitution in human TS increases the Kd for FdUMP binding into a covalent ITC (5, 23). In the current study, we show that FdUMP binding into a binary complex, i.e., in the absence of CH2H4PteGlu, occurs with virtually identical Kd values for both the Tyr33 and the His33 enzyme forms. Interestingly, the rate constants describing FdUMP association and dissociation (i.e., kon and koff , respectively) did differ between the two enzyme forms, in that the Tyr33 r His substitution caused a two- to threefold reduction in both kon and koff . However, since the magnitude of the reductions was similar for both rate constants, the overall Kd was unaffected. Thus, the impact of the Tyr33 r His substitution on overall FdUMP binding to TS is observed only in ternary complexes, i.e., in the presence of CH2H4PteGlu. Recent X-ray studies of human TS have indicated that Tyr33, which is located within an amphipathic a-helix, donates a hydrogen bond to the main-chain carbonyl of Met219, which is located at the base of a second a-helix (33). The latter helix forms part of the enzyme’s active site cavity and contains several residues that participate in ligand binding (15–19, 33). This is a conserved feature of the TS molecules whose crystal structures have been determined (15–19). It is possible that the absence of this hydrogen bond in the His33 enzyme underlies the effects of the Tyr33 r His substitution on TS function; however, the fact that the Phe33 form, which also lacks the hydrogen bond, exhibits properties that are essentially indistinguishable from the wild-type enzyme (5, 23; Table I), makes this

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unlikely. Schiffer et al. (33) have suggested that the positive charge on the imidazole ring of His33 may attract the negatively charged side chain of Asp218, resulting in conformational perturbation of the active site. Crystal structures of the His33 enzyme are consistent with this suggestion.6 The mechanisms underlying the differential impact of the His33 substitution on binary and ternary complexes are not known. It may be that binding of CH2H4PteGlu to the enzyme elicits a conformational shift that alters the relative position of residue 33; for the His33 enzyme, this shift may result in a microenvironment in which the imidazole ring can elicit changes in the kinetics of FdUMP binding. X-ray studies have indeed shown that binding of folate cosubstrate on its own to TS induces conformational changes in the polypeptide (3, 34). However, it has also been shown that bound dUMP exists in similar conformations and is in contact with the same amino acid side-chains in both binary and ternary complexes (3, 16, 19). The differences, if any, between the microenvironments of FdUMP bound in binary vs ternary complexes are not completely known. The results of our experiments may relate to the negative cooperativity model invoked as an explanation for the properties of ternary complexes of the His33 enzyme (5). Several studies, including the current one, have indicated that the stoichiometry of FdUMP binding into a covalent binary complex is 0.5–0.7/dimer (24–27); thus, the second subunit may be relatively incapable of covalent nucleotide binding in the absence of CH2H4PteGlu. It is possible that this reflects an intrinsic lack of availability of the second site. However, the fact that the two subunits of ligand-free TS are identical in conformation (3, 15–19) makes it likely that the absence of FdUMP binding into a binary covalent complex at the second subunit reflects strong negative cooperativity, where covalent binding at the first subunit effectively precludes binding at the second. Our finding of similar Kd values for binary complexes between FdUMP and the Tyr33 or His33 forms implies that the Tyr33 r His substitution does not impact on overall FdUMP affinity under conditions where only one of the two subunits is available for covalent ligand binding. This is in sharp contrast to the situation with the ITC, the formation of which occurs at both subunits of the dimer (5–7). For such complexes, the Tyr33 r His substitution increases the Kd for FdUMP binding at the second, but not the first, subunit (5). Thus, the Tyr33 r His substitution does not appear to impact upon overall FdUMP affinity unless both subunits are participating in covalent ligand binding. This implies that subunit interactions in TS are of potential importance in ligand binding, and provides indirect support for a cooperativity model (5). 6

G. Sayer, personal communication.

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Analysis of the properties of a number of mutant TS molecules containing amino acid substitutions at position 33 (Table I, Fig. 4) strongly indicates that maximal TS accumulation and function requires a bulky, hydrophobic amino acid at this site. Several of the mutants (i.e., His33, Leu33, Ile33, Val33, and Trp33) retain catalytic function, although they exhibit kcat values that are only 10–50% of the those exhibited by the wild-type and Phe33 enzymes (Table I). Since the side chain of residue 33 lies on the hydrophobic face of an amphipathic a-helix (15–19, 33), it is likely that low levels of enzyme expressed by mutants with charged or polar amino acids at residue 33 (i.e., Ala33, Ser33, Asp33, Asn33, Lys33, and Gly33) (Fig. 4) are due to high enzyme instability, as a consequence of extensive perturbations in TS structure. The fact that substitutions at residue 33 elicit such profound effects upon TS accumulation and function may explain the high degree of evolutionary conservation at this site. Indeed, among the 29 TS molecules that have been sequenced, 28 have tyrosine at the position analagous to residue 33 of the human enzyme, while one has phenyalanine (3). The reduced accumulation and catalytic efficiencies exhibited by TS polypeptides with amino acids other than tyrosine or phenylalanine at residue 33 are likely to be physiologically disadvantageous and may be selected against during evolution. ACKNOWLEDGMENTS The authors thank Dr. Sondra Berger and Ms. Lynette Washington for providing the hybridoma cell line, along with technical advice on its use in Western blot analysis.

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