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Irreversible inhibition of thymidylate synthetase by 5-formyl-2′-deoxyuridylic acid
Vol. 46, No. 3, 1972
BIOCHEMICALAND BIOPHYSICAL RESEARCH COMMUNICATIONS
IRREVERSIBLE INHIBITION OF THYMIDYLATE SYNTHETASE BY 5-FORMYL-2'-DEOXYURIDYL!C ACID
Daniel V. Santi and Ted T. Sakai Department of Chemistry University of California Santa Barbara, California 93106
R e c e i v e d D e c e m b e r 30, 1971 SUMMARY: The thymidylate synthetases from E. coli B and L. casei are rapidly and irreversibly inactivated upon incubation with 5-formyl-2'-deoxyuridylic acid. Evidence is presented which demonstrates that the inactivation proceeds via initial complexation of the inhibitor at the active site. From chemical considerations, it is suggested that a Schiff base is formed between the inhibitor and a primary amino group of the enzyme. Implications with regard to catalysis are discussed. Thymidylate synthetase catalyzes the reduetive methylation of 2'-deoxyuridylic acid (dUMP) to give thymidylic acid (TMP) with concomitant conversion of the cofactor 5,10-methylenetetrahydrofolic acid (5,10-CH2FAH4) to 7~8-dihydrofolic acid (7~8-FAH2).
5-Formyl-2'-deoxyuridylic acid (FOdUMP) has recently been
shown to be a potent competitive inhibitor of the thymidylate synthetase from E. coli B (i).
We now report that incubation of low concentrations of FOdUMP
with the enzymes from E. coli and dichloromethotrexate(DCM)-resistant L. casei results in an irreversible inactivation which is mediated through the reversible complex.
Chemical considerations lead us to surmise that the inhibitor reacts
with a primary amino group at the active site to form a Schiff base. Materials and Methods The previously described (i) preparation of thymidylate synthetase from E. eoli B was purified further by hydroxylapatite chromatography using a linear gradient from 0.01 M to 0.15 M potassium phosphate (pH 6.8) containing 20mM 2-mercaptoethanol, i mM_MgCI2, and i0@ glycerol. 720 nmoles ~P/min/mg at 25 °.
The final preparation catalyzed
The enzyme from DCM-resistant L. easel (2) was
prepared by the method of Leafy and Kisliuk (3) and produced 2000 nmoles ~MP/ min/mg at 25 ° . All assays described were conducted at 25 °. velocity assay contained O.08mM_dUMP,
The standard initial
0.12 mMD~L-FAH4, 6.5 mM formaldehyde,
25 n~M_MgCI2, i mMEDTA, 75 mM 2-mereaptoethanol, and 50 mMN-methylmorpholine'HCl buffer (pH 7.40) in a total volume of i.i ml.
The reaction was initiated by the
addition of a limiting amount (i-2 units) of enzyme and monitored spectrophotometrically (4); duMP was omitted for controls.
1320 Copyright © 1972, by Academic Press, Inc.
A unit is defined as that amount
Vol. 46, No. 3, 1972
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
of enzyme which will catalyze the formation of i nmole/TMP/min under the standard assay conditions.
To obtain double reciprocal plots after preincubation with
FOdUMP, reactions were performed in cuvettes containing all components except dUMP.
After 15 minutes, the reaction was initiated by the addition of the
appropriate amount of dUMP.
For inactivation and protection experiments, incuba-
tion mixtures contained 6 mM N-methylmorpholine. HCl buffer (pH 7.40), 3 mM MgCI2, 0.12 raM_EDTA, 9 mM 2-mercaptoethanol, ca. 25 units of thymidylate synthetase/ml (ca. 0.17 ~M), and the specified amounts of FOdUMP or protecting agents.
At
appropriate intervals, 50 ~i aliquots were removed and assayed. Results FOdUMP is a competitive inhibitor with respect to dUMP with K. = 1.3 x 10 -8 l
M and 1.2 x 10-8 M for the thymidylate synthetases from E. coli B (i) and L. easel (Figure i), respectively.
After incubation of the enzymes with FOdUMP for ,
J
,/
,;o
,~o
~..2 .c=
%1 ~o
1/{dUMP),mM"
Figure l: Double reciprocal plots of data for L. casei enzyme for FOdUMP at varying dUMP; inhibitor concentrations are 0 ( O ), 1.25 x IO-SM ( A ) , and 2.5 x lO -8 M ( [] ). Velocities are expressed in nmoles/min. --
i
5
i
i
A
4 / A
/
2 .f@-@
0 --510
100 5J0 l/[dUMP], m_M-t
I
__
100
Figure 2: Double reciprocal plots of data for E. coli enzyme (A) and L. casei enzyme (B) for FOdDMP at varying dUMP after 15 rain. preincubation; inhibitor concentrations are 0 ( @ ) , 1.5 x l O - S M ( ~ ) , and 3.0 x lO-S • ( A ) .
1321
Vol. 46, No. 3, 1972
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
15 minutes in the presence of all reaction components except dUMP, the inhibition becomes non-competitive with respect to dUMP (Figure 2). The stability of these enzymes in the absence and presence of FOdUMP, as well as agents which compete with FOdUMP for its binding site is shown in Figure 3.
The E. coli enzyme is rather unstable at 25°~ showing a first-order loss of
activity with t~ = 124 min (Figure 3A).
The enzyme is stabilized significantly
by dDMP (tl = 1030 min), but not 5,10-CHeFAH4 (tA = 113 min).
In this regard,
we have recently observed that FAH~ prepared by catalytic hydrogenation contains a contaminant which rapidly inactivates the thymidylate synthetase from L. casei. It is not known at this time to what extent the aforementioned inactivation of the E. coli enzyme is attributable to this contaminant.
In other experiments
described, the FAH4 utilized was freed from this impurity by DEAE chromatography. In the presence of 1.5 x 10-7 M F0dUMP, the rate of inactivation is increased about two-fold and 15-fold over that observed in the absence and presence of dDMP~ respectively.
The inactivation may be retarded by HMdUMP [a competitive
inhibitor with respect to dUMP (i)] but not 5,10-CH2FAH4. As shown in Figure 3B, the L. case{ enzyme is considerably more stable, and shows no loss of activity at 25 ° after as long as 50 minutes in the absence 100
B
-
>.. I,,,,, I*,,-
U
I
l 40
I
I
/
20
80
Time
40
(min)
Figure >: (A) Activity of E. coli enzyme as a function of time for enzyme (E) , ~ + 6 x lo-S M dump ( ] & ~ + 5 X iO-4 ~ 5 , 1 0 - C ~ F m % ( A ) , ~ + 1.5 x ~0~ ~ FOduMP ( • ),--~ + i.~ x 10 7 M FOduMP + Y.o x lO ~ M ~ d u M P ( [] ). (B) ~ctivity of L. easei enzyme wit[ for enzyme (E) alone--( • ), E + 5 x IO_-4M 5 , 1 0 - C ½ F A ~ + 3 ~_ lO -6 ~_ FOdUMP ( • ), v + 5 x i0 -~ M 5,10-C~2FA~ + 3 x lo 6M FOdUMP + 1.4 x iO 4 M 19~dUMP (/k); E ÷ 5 x lO -4 M 5,YO-CH2FAH4, E + 1.4 x i0 ~M_ }~4dUMP, and E + 1.4 x 10 -4 M_ }~4dDMP + 5 x 10 -4 M_ 5,10-CHsFAH4 are identical with base line (E alone). "
1322
Vol. 46, No. 3, 1972
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
or presence of dUMP, 5,10-CH2FAH~, or HMdUMP and 5,10-CH2FAH4.
In apparent
contrast to the E. coli enzym% FOduMP causes negligible inactivation when incubated alone with the enzyme.
However, in the presence of 5,10-CH2FAH~, FOdUMP
causes a rapid loss of activity which is first order with respect to remaining enzyme.
As with the E. coli enzyme, protection against FOdUMP inactivation is
afforded by HMdUMP, a competitive inhibitor with respect to dDMP (Ki = 2.1 x i0 -z ~).
l-Methyl-5-formyluracil (5) does not inhibit or inactivate the enzyme
at concentrations up to 3 x 10-4 M. Discussion The change from competitive to noncompetitive inhibition kinetics which occurs upon incubation of FOduMP with E. coli thymidylate synthetase is suggestive of active site directed irreversible inactivation; this is also supported by the protection afforded against the FOduMP mediated inhibition by the competitive inhibitor UMduMP.
Unfortunately, further kinetic interpretation is
complicated by the inherent instability of the E. coli enzyme.
For example, the
rate of inactivation by FOdUMP is twice faster than the spontaneous rate in the absence of dUMP, but 15-fold faster than in the presence of dUMP.
Since com-
pounds which complex to the dUMP binding site (dUMP~ HMdUMP) apparently protect the enzyme against spontaneous activation, it is likely that FOdUMP also affords similar protection.
Thus, comparison of the rate of inactivation by FOdUMP to
that obtained in the absence of protecting agents provides only a minimum value for the effectiveness of this compound.
To avoid these complications~ efforts
were turned to the thymidylate synthetase obtained from DCM-resistant L. casei which is stable under conditions of the inactivation experiments. As with the E. coli enzyme, conversion of competitive to noncompetitive inhibition patterns upon incubation of FOdUMP with the L. casei thymidylate synthetase and all reaction components except dUMP suggests irreversible inhibition (Figure 2B).
The experiments presented in Figure 3B show that, in the presence
of 5,10-CH2FAH4~ FOdUMP causes inactivation whereas the enzyme is stable in the absence of either or both components.
The direct participation of the 5-formyl
group of FOdUMP in the inactivation is evidenced by the inertness of the enzyme toward 5,10-CH2FAH4, or 5~IO-CH2FAH~ and HMdUMP; the latter analog binds reversibly to the enzyme, and differs from FOdUMP only in the substitution of hydroxymethyl for the 5-formyl group.
That this inactivation occurs after complexation
of FOdUMP at the active site is demonstrated by four lines of evidence:
(i) At
the low concentrations of FOdUMP and enzyme used, random bimolecular reaction of the aldehyde group should not occur at an appreciable rate;
(2) l-Methyl-5-
formyl~racil, an aldehyde of comparable reactivity, which does not reversibly bind to the enzymes, does not cause inactivation.
It is also noted that enzyme
assays are performed routinely at high formaldehyde concentrations without
1323
Vol. 46, No. 3, 1972
BIOCHEMICALAND BIOPHYSICAL RESEARCH COMMUNICATIONS
deleterious effects.
(3)
Protection against inactivation by FOdUMP is afforded
by reagents which compete reversibly for its binding site. contrast to the E. coli enzym%
(4)
In apparent
thymidylate synthetase from L. casei is stable
toward FOdUMP if 5,10-CH2FAH 4 is not present. results in rapid inactivation.
However, addition of the cofactor
In this regard, it is of interest to note that
substrate binding to thymidylate synthetase from Ascites cells has been proposed to proceed by an ordered sequence in which 5,10-CH2FAH4 must bind prior to dUMp
(7). Since the only functional group present in proteins which may form a rela-
tively stable covalent linkage with aldehydes is a primary smine, it is most reasonable to suggest that after reversible complexation, FOdUMP forms a Schiff base with the amino group of a lysine residue or the N-terminal amino acid of the enzyme (equation i).
In this regard, i% is noted that 35 of the some 537 amino
acid residues of thymidylate synthetase from amethopterin-resistant L. casei analyze as lysine (6).
dUMP-5-CHO
+
HeN-Enz ~
"~ duMP-5-CHO"'H2N-Enz
-HeO ~ dUMP-5-CH=N-Enz
(i)
K. l
Although the presence of a primary amino group in this region of the enzyme could be considered fortuitous, catalytic roles may be envisioned which are in accord with mechanistic proposals forwarded in this laboratory.
Model studies
have led to the suggestion that the enzymic reaction requires the addition of a nucleophile to the 6-position of the heterocycle (8,9) in order to activate the 5-carbon toward electrophilic attack by proton, leading to the observed 5-~ exchange (i0), or formaldehyde (as 5,10-CH2FA~ or equivalent species) to give an intermediate which breaks down to FAH2 and TMP.
The chemical counterparts
may be facilitated by protonation of the 4-keto group of the pyrimidine or by bases which aid in the abstraction of the 5-hydrogen subsequent to addition (11,12).
Depending on the state of ionization~ and allowing for minor con-
formational
changes of the enzyme during the course of reaction, an amino group
in the vicinity of the 5-position of dUMP in the enzyme-substrate complex might function in either of these catalytic modes.
It is also conceivable that a
primary amino group could act as a carrier of formaldehyde from 5,10-CH2FAH4 to dUMP~ and may represent the elusive "methylated" enzyme sought by earlier workers (13).
Further studies directed at characterization of the reactive amino
group and possible roles in catalysis are in progress. Acknowledgements: This work was supported by Public Health Service Research Grant No. CA-I0499 from the National Cancer Institute and an NIX Predoctoral Fellowship to T.T.S. We are indebted to Drs. R. L. Kisliuk, R. P. Leafy, and S. Rothman for their aid in the preparation of the L. easei thymidylate synthetase.
1324
Vol. 46, No. 3, 1972
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
References i. 2. 3. 4. 5. 6. 7. 8. 9. io. !i. 12. 13.
Santi, D. V., and Sakai, T. T. (1971), Biochem. Biophys. Res. Commun., 813. Crusberg, T. C., Leary~ R., and Kisliuk, R. L. (1970), J. Biol. Chem., 24~, 5 292. Leafy, R., and Kisliuk~ R. L. (1971), Prep. Biochem., i_~ 47. Wahba, A. J., and Friedkin, M. (1961), J. Biol. Chem., ~ PC ii. Saka±, T. T., Pogolotti, A. L., Jr., and Santi, D. V. (1968), J. Heterocyel. Chem., ~ 849. Huennekens, F. M., personal communication. Reyes, Santi, Santi, 9o3. Lomax, Santi, Santi, Wahba,
P., and Heidelberger, C. (1965), Mol. Pharmacol., i_~ 14. D. V., and Brewer, C. F. (1968), J. Amer° Chem. Soc., 90. 6236. D. V., Brewer, C. F., and Farber, D. (1970), J. Heterocycl. Chem.~ L M. D. D. A.
I. S., and Greenberg, G. R. (1967), J. Biol. Chem., 242., 1302. V., and Brewer~ C. F.~ unpublished results. V., and Maley, J. R., unpublished results. J., and Friedkin, M. (1962), J. Biol. Chem., 237, 3794.
1325
BIOCHEMICALAND BIOPHYSICAL RESEARCH COMMUNICATIONS
IRREVERSIBLE INHIBITION OF THYMIDYLATE SYNTHETASE BY 5-FORMYL-2'-DEOXYURIDYL!C ACID
Daniel V. Santi and Ted T. Sakai Department of Chemistry University of California Santa Barbara, California 93106
R e c e i v e d D e c e m b e r 30, 1971 SUMMARY: The thymidylate synthetases from E. coli B and L. casei are rapidly and irreversibly inactivated upon incubation with 5-formyl-2'-deoxyuridylic acid. Evidence is presented which demonstrates that the inactivation proceeds via initial complexation of the inhibitor at the active site. From chemical considerations, it is suggested that a Schiff base is formed between the inhibitor and a primary amino group of the enzyme. Implications with regard to catalysis are discussed. Thymidylate synthetase catalyzes the reduetive methylation of 2'-deoxyuridylic acid (dUMP) to give thymidylic acid (TMP) with concomitant conversion of the cofactor 5,10-methylenetetrahydrofolic acid (5,10-CH2FAH4) to 7~8-dihydrofolic acid (7~8-FAH2).
5-Formyl-2'-deoxyuridylic acid (FOdUMP) has recently been
shown to be a potent competitive inhibitor of the thymidylate synthetase from E. coli B (i).
We now report that incubation of low concentrations of FOdUMP
with the enzymes from E. coli and dichloromethotrexate(DCM)-resistant L. casei results in an irreversible inactivation which is mediated through the reversible complex.
Chemical considerations lead us to surmise that the inhibitor reacts
with a primary amino group at the active site to form a Schiff base. Materials and Methods The previously described (i) preparation of thymidylate synthetase from E. eoli B was purified further by hydroxylapatite chromatography using a linear gradient from 0.01 M to 0.15 M potassium phosphate (pH 6.8) containing 20mM 2-mercaptoethanol, i mM_MgCI2, and i0@ glycerol. 720 nmoles ~P/min/mg at 25 °.
The final preparation catalyzed
The enzyme from DCM-resistant L. easel (2) was
prepared by the method of Leafy and Kisliuk (3) and produced 2000 nmoles ~MP/ min/mg at 25 ° . All assays described were conducted at 25 °. velocity assay contained O.08mM_dUMP,
The standard initial
0.12 mMD~L-FAH4, 6.5 mM formaldehyde,
25 n~M_MgCI2, i mMEDTA, 75 mM 2-mereaptoethanol, and 50 mMN-methylmorpholine'HCl buffer (pH 7.40) in a total volume of i.i ml.
The reaction was initiated by the
addition of a limiting amount (i-2 units) of enzyme and monitored spectrophotometrically (4); duMP was omitted for controls.
1320 Copyright © 1972, by Academic Press, Inc.
A unit is defined as that amount
Vol. 46, No. 3, 1972
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
of enzyme which will catalyze the formation of i nmole/TMP/min under the standard assay conditions.
To obtain double reciprocal plots after preincubation with
FOdUMP, reactions were performed in cuvettes containing all components except dUMP.
After 15 minutes, the reaction was initiated by the addition of the
appropriate amount of dUMP.
For inactivation and protection experiments, incuba-
tion mixtures contained 6 mM N-methylmorpholine. HCl buffer (pH 7.40), 3 mM MgCI2, 0.12 raM_EDTA, 9 mM 2-mercaptoethanol, ca. 25 units of thymidylate synthetase/ml (ca. 0.17 ~M), and the specified amounts of FOdUMP or protecting agents.
At
appropriate intervals, 50 ~i aliquots were removed and assayed. Results FOdUMP is a competitive inhibitor with respect to dUMP with K. = 1.3 x 10 -8 l
M and 1.2 x 10-8 M for the thymidylate synthetases from E. coli B (i) and L. easel (Figure i), respectively.
After incubation of the enzymes with FOdUMP for ,
J
,/
,;o
,~o
~..2 .c=
%1 ~o
1/{dUMP),mM"
Figure l: Double reciprocal plots of data for L. casei enzyme for FOdUMP at varying dUMP; inhibitor concentrations are 0 ( O ), 1.25 x IO-SM ( A ) , and 2.5 x lO -8 M ( [] ). Velocities are expressed in nmoles/min. --
i
5
i
i
A
4 / A
/
2 .f@-@
0 --510
100 5J0 l/[dUMP], m_M-t
I
__
100
Figure 2: Double reciprocal plots of data for E. coli enzyme (A) and L. casei enzyme (B) for FOdDMP at varying dUMP after 15 rain. preincubation; inhibitor concentrations are 0 ( @ ) , 1.5 x l O - S M ( ~ ) , and 3.0 x lO-S • ( A ) .
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Vol. 46, No. 3, 1972
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
15 minutes in the presence of all reaction components except dUMP, the inhibition becomes non-competitive with respect to dUMP (Figure 2). The stability of these enzymes in the absence and presence of FOdUMP, as well as agents which compete with FOdUMP for its binding site is shown in Figure 3.
The E. coli enzyme is rather unstable at 25°~ showing a first-order loss of
activity with t~ = 124 min (Figure 3A).
The enzyme is stabilized significantly
by dDMP (tl = 1030 min), but not 5,10-CHeFAH4 (tA = 113 min).
In this regard,
we have recently observed that FAH~ prepared by catalytic hydrogenation contains a contaminant which rapidly inactivates the thymidylate synthetase from L. casei. It is not known at this time to what extent the aforementioned inactivation of the E. coli enzyme is attributable to this contaminant.
In other experiments
described, the FAH4 utilized was freed from this impurity by DEAE chromatography. In the presence of 1.5 x 10-7 M F0dUMP, the rate of inactivation is increased about two-fold and 15-fold over that observed in the absence and presence of dDMP~ respectively.
The inactivation may be retarded by HMdUMP [a competitive
inhibitor with respect to dUMP (i)] but not 5,10-CH2FAH4. As shown in Figure 3B, the L. case{ enzyme is considerably more stable, and shows no loss of activity at 25 ° after as long as 50 minutes in the absence 100
B
-
>.. I,,,,, I*,,-
U
I
l 40
I
I
/
20
80
Time
40
(min)
Figure >: (A) Activity of E. coli enzyme as a function of time for enzyme (E) , ~ + 6 x lo-S M dump ( ] & ~ + 5 X iO-4 ~ 5 , 1 0 - C ~ F m % ( A ) , ~ + 1.5 x ~0~ ~ FOduMP ( • ),--~ + i.~ x 10 7 M FOduMP + Y.o x lO ~ M ~ d u M P ( [] ). (B) ~ctivity of L. easei enzyme wit[ for enzyme (E) alone--( • ), E + 5 x IO_-4M 5 , 1 0 - C ½ F A ~ + 3 ~_ lO -6 ~_ FOdUMP ( • ), v + 5 x i0 -~ M 5,10-C~2FA~ + 3 x lo 6M FOdUMP + 1.4 x iO 4 M 19~dUMP (/k); E ÷ 5 x lO -4 M 5,YO-CH2FAH4, E + 1.4 x i0 ~M_ }~4dUMP, and E + 1.4 x 10 -4 M_ }~4dDMP + 5 x 10 -4 M_ 5,10-CHsFAH4 are identical with base line (E alone). "
1322
Vol. 46, No. 3, 1972
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
or presence of dUMP, 5,10-CH2FAH~, or HMdUMP and 5,10-CH2FAH4.
In apparent
contrast to the E. coli enzym% FOduMP causes negligible inactivation when incubated alone with the enzyme.
However, in the presence of 5,10-CH2FAH~, FOdUMP
causes a rapid loss of activity which is first order with respect to remaining enzyme.
As with the E. coli enzyme, protection against FOdUMP inactivation is
afforded by HMdUMP, a competitive inhibitor with respect to dDMP (Ki = 2.1 x i0 -z ~).
l-Methyl-5-formyluracil (5) does not inhibit or inactivate the enzyme
at concentrations up to 3 x 10-4 M. Discussion The change from competitive to noncompetitive inhibition kinetics which occurs upon incubation of FOduMP with E. coli thymidylate synthetase is suggestive of active site directed irreversible inactivation; this is also supported by the protection afforded against the FOduMP mediated inhibition by the competitive inhibitor UMduMP.
Unfortunately, further kinetic interpretation is
complicated by the inherent instability of the E. coli enzyme.
For example, the
rate of inactivation by FOdUMP is twice faster than the spontaneous rate in the absence of dUMP, but 15-fold faster than in the presence of dUMP.
Since com-
pounds which complex to the dUMP binding site (dUMP~ HMdUMP) apparently protect the enzyme against spontaneous activation, it is likely that FOdUMP also affords similar protection.
Thus, comparison of the rate of inactivation by FOdUMP to
that obtained in the absence of protecting agents provides only a minimum value for the effectiveness of this compound.
To avoid these complications~ efforts
were turned to the thymidylate synthetase obtained from DCM-resistant L. casei which is stable under conditions of the inactivation experiments. As with the E. coli enzyme, conversion of competitive to noncompetitive inhibition patterns upon incubation of FOdUMP with the L. casei thymidylate synthetase and all reaction components except dUMP suggests irreversible inhibition (Figure 2B).
The experiments presented in Figure 3B show that, in the presence
of 5,10-CH2FAH4~ FOdUMP causes inactivation whereas the enzyme is stable in the absence of either or both components.
The direct participation of the 5-formyl
group of FOdUMP in the inactivation is evidenced by the inertness of the enzyme toward 5,10-CH2FAH4, or 5~IO-CH2FAH~ and HMdUMP; the latter analog binds reversibly to the enzyme, and differs from FOdUMP only in the substitution of hydroxymethyl for the 5-formyl group.
That this inactivation occurs after complexation
of FOdUMP at the active site is demonstrated by four lines of evidence:
(i) At
the low concentrations of FOdUMP and enzyme used, random bimolecular reaction of the aldehyde group should not occur at an appreciable rate;
(2) l-Methyl-5-
formyl~racil, an aldehyde of comparable reactivity, which does not reversibly bind to the enzymes, does not cause inactivation.
It is also noted that enzyme
assays are performed routinely at high formaldehyde concentrations without
1323
Vol. 46, No. 3, 1972
BIOCHEMICALAND BIOPHYSICAL RESEARCH COMMUNICATIONS
deleterious effects.
(3)
Protection against inactivation by FOdUMP is afforded
by reagents which compete reversibly for its binding site. contrast to the E. coli enzym%
(4)
In apparent
thymidylate synthetase from L. casei is stable
toward FOdUMP if 5,10-CH2FAH 4 is not present. results in rapid inactivation.
However, addition of the cofactor
In this regard, it is of interest to note that
substrate binding to thymidylate synthetase from Ascites cells has been proposed to proceed by an ordered sequence in which 5,10-CH2FAH4 must bind prior to dUMp
(7). Since the only functional group present in proteins which may form a rela-
tively stable covalent linkage with aldehydes is a primary smine, it is most reasonable to suggest that after reversible complexation, FOdUMP forms a Schiff base with the amino group of a lysine residue or the N-terminal amino acid of the enzyme (equation i).
In this regard, i% is noted that 35 of the some 537 amino
acid residues of thymidylate synthetase from amethopterin-resistant L. casei analyze as lysine (6).
dUMP-5-CHO
+
HeN-Enz ~
"~ duMP-5-CHO"'H2N-Enz
-HeO ~ dUMP-5-CH=N-Enz
(i)
K. l
Although the presence of a primary amino group in this region of the enzyme could be considered fortuitous, catalytic roles may be envisioned which are in accord with mechanistic proposals forwarded in this laboratory.
Model studies
have led to the suggestion that the enzymic reaction requires the addition of a nucleophile to the 6-position of the heterocycle (8,9) in order to activate the 5-carbon toward electrophilic attack by proton, leading to the observed 5-~ exchange (i0), or formaldehyde (as 5,10-CH2FA~ or equivalent species) to give an intermediate which breaks down to FAH2 and TMP.
The chemical counterparts
may be facilitated by protonation of the 4-keto group of the pyrimidine or by bases which aid in the abstraction of the 5-hydrogen subsequent to addition (11,12).
Depending on the state of ionization~ and allowing for minor con-
formational
changes of the enzyme during the course of reaction, an amino group
in the vicinity of the 5-position of dUMP in the enzyme-substrate complex might function in either of these catalytic modes.
It is also conceivable that a
primary amino group could act as a carrier of formaldehyde from 5,10-CH2FAH4 to dUMP~ and may represent the elusive "methylated" enzyme sought by earlier workers (13).
Further studies directed at characterization of the reactive amino
group and possible roles in catalysis are in progress. Acknowledgements: This work was supported by Public Health Service Research Grant No. CA-I0499 from the National Cancer Institute and an NIX Predoctoral Fellowship to T.T.S. We are indebted to Drs. R. L. Kisliuk, R. P. Leafy, and S. Rothman for their aid in the preparation of the L. easei thymidylate synthetase.
1324
Vol. 46, No. 3, 1972
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
References i. 2. 3. 4. 5. 6. 7. 8. 9. io. !i. 12. 13.
Santi, D. V., and Sakai, T. T. (1971), Biochem. Biophys. Res. Commun., 813. Crusberg, T. C., Leary~ R., and Kisliuk, R. L. (1970), J. Biol. Chem., 24~, 5 292. Leafy, R., and Kisliuk~ R. L. (1971), Prep. Biochem., i_~ 47. Wahba, A. J., and Friedkin, M. (1961), J. Biol. Chem., ~ PC ii. Saka±, T. T., Pogolotti, A. L., Jr., and Santi, D. V. (1968), J. Heterocyel. Chem., ~ 849. Huennekens, F. M., personal communication. Reyes, Santi, Santi, 9o3. Lomax, Santi, Santi, Wahba,
P., and Heidelberger, C. (1965), Mol. Pharmacol., i_~ 14. D. V., and Brewer, C. F. (1968), J. Amer° Chem. Soc., 90. 6236. D. V., Brewer, C. F., and Farber, D. (1970), J. Heterocycl. Chem.~ L M. D. D. A.
I. S., and Greenberg, G. R. (1967), J. Biol. Chem., 242., 1302. V., and Brewer~ C. F.~ unpublished results. V., and Maley, J. R., unpublished results. J., and Friedkin, M. (1962), J. Biol. Chem., 237, 3794.
1325