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Modification of Serratia marcescens anthranilate synthase with pyridoxal 5′-phosphate
ARCHIVES
Vol.
219,
OF BIOCHEMISTRY
No.
AND
1, November,
pp.
Modification
BIOPHYSICS 121-127, 1982
of Serratia marcescens Anthranilate with Pyridoxal S-Phosphate’ STANLEY
Department
BOWER
of Biochemistry,
AND
Purdue
HOWARD
Uniuersity,
Received
June
West
Synthase
ZALKIN2 Lafayette,
Indiana
47907
10, 1982
The glutamine-dependent activity of Serrutia marcescens anthranilate synthase was inactivated by pyridoxal 5’-phosphate and sodium cyanide. The reaction was specific in that the ammonia-dependent activity of the enzyme was unaffected. The inactivation was stable to dilution or dialysis hut was reversed by dithiothreitol. The enzyme contains dissimilar subunits designated anthranilate synthase components I (AS I) and II (AS II). Incorporation of [14C]NaCN demonstrates that modification was limited to one to two residues per AS 1. AS II protomer. An active site cysteine is involved in the glutamine-dependent activity. Modification by pyridoxal 5’-phosphate and NaCN blocked affinity labeling of the active site cysteine by the glutamine analog 6-diazo-5-0x0-b norleucine and reduced alkylation of the active site cysteine by iodoacetamide. These results suggest modification is at the glutamine active site. Initial modification by iodoacetamide did not prevent pyridoxal 5’-phosphate-dependent incorporation of 14CN showing that the pyridoxal 5’-phosphate modification did not involve the essential cysteinyl residue. These results suggest that modification of a lysyl residue in the glutamine active site of anthranilate synthase reduces the reactivity of the essential cysteinyl residue resulting in the loss of the amidotransferase activity.
Anthranilate synthase catalyzes the first reaction of tryptophan biosynthesis. The enzyme from enteric bacteria is an oligomerit protein containing dissimilar suhunits designated AS I and AS II.3 AS I catalyzes the reaction shown in Eq. [l]:
AS II provides glutamine amidotransferase function for the oligomeric enzyme complex (Eq. [Z]). Chorismate Anthranilate
M$+
Chorismate
+ glutamine + pyruvate
+ glutamate
+ NH3 Anthranilate
Mg*+
-
+ pyruvate
[2]
Anthranilate synthase is one of the best understood of the many glutamine amidotransferases in prokaryotic and eukaryotic cells (1, 2) and has served as a model for understanding more complex enzymes of this type (3). The amino acid sequences of the AS I subunit from Escherichia coli and Salmonella typhimurium (4) have been deduced from the DNA sequences. Amino acid sequences of AS IT subunits from several bacteria have been obtained by protein and DNA sequencing (5-7). Amino acid residues have been identified which are
[l]
’ This work was supported hy Public Health Service Grant GM 24658 from the National Institute of General Medical Sciences. This is Journal Paper Number 9052 from the Purdue Agricultural Experiment Station. ‘Author to whom correspondence should be addressed. ’ Abbreviations used: AS I, anthranilate synthase component I; AS II, anthranilate synthase component II; pyridoxal-P, pyridoxal %phosphate; DON, 6-diazo5-oxo-1,.norleucine; Hepes, N-2-hydroxyethylpiperazinc-l\i’-2.ethane sulfonic acid. 121
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rights
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in any form
reserved.
122
BOWER
AND
essential to the function of each subunit (7, 8). Single arginyl, histidyl, and cysteinyl residues in AS I were found to be essential for the NH,-dependent reaction (8). The roles of these residues are not clearly understood but they appear to be involved with binding of chorismate and its conversion to anthranilate plus pyruvate. Glutamine amidotransferase function requires an AS II active site cysteinyl residue (7). The role of this active site cysteinyl residue in activation of glutamine and amide transfer was first worked out for anthranilate synthase (9) and appears to be similar for other glutamine amidotransferases (10). In this report we provide the first evidence for a second residue essential for the glutamine amide transfer function. This amino acid was modified by treatment of anthranilate synthase with pyridoxal-P. The essential residue exhibits properties of an unique lysyl residue. MATERIALS
AND
METHODS
Materials. Anthranilate synthase was purified from S. marcescens (ATCC 27143) by J. Yun Tso (7). [6‘%]DON was prepared by the method of Hartman (11) from N-trifluoroacetyl-L-glutamic-y-acid chloride-a-methyl ester which was a generous gift from Dr. John S. Holcenberg, Medical College of Wisconsin. Iodo[l-%]acetamide and [“C]NaCN were from New England Nuclear. Most other chemicals were commercial products of the highest purity available and were used without further purification. Methods. Anthranilate synthase was assayed as previously described (12, 13). Inactivations were performed in 50 mM potassium phosphate pH 7.4 containing 0.1 mM EDTA. The reactions were conducted at room temperature and were initiated by the addition of pyridoxal-P, DON, or iodoacetamide. Aliquots of the reaction mixture were diluted loo-fold into the standard assay mixture. In some cases aliquots of up to 100 ~1 were rapidly desalted by centrifugation gel filtration (14). Excess reagents were removed from preparative scale modifications by dialysis. When inactivation was by pyridoxal-P and NaBH,, anthranilate synthase was incubated in 0.25 ml of 50 mM potassium phosphate pH 7.4, 0.1 mM EDTA and pyridoxal-P. After 20 min 20 ~1 of n-octanol was added followed by 0.25 ml of 0.1 M NaBH,. After a 5-min incubation at 4°C samples were dialyzed against 50 mM potassium phosphate pH 7.4, 0.1 mM EDTA, and 0.2 mM dithiothreitol. Activity was determined by the standard assay. Protein con-
ZALKIN centration and the number of lysyl residues modified were determined by amino acid analysis. Second-order rate constants for enzyme inactivation were determined from the slope of plots of pseudo-first-order rate constants versus reagent concentration. Data were fit by linear regression. Anthranilate synthase concentrations were routinely determined by absorbance at 278 nm using an extinction coehicient, &” = 0.658,’ which was confirmed by amino acid analysis. Amino acid analyses were performed with a Durrum D 500 amino acid analyzer. RESULTS
Inactivation
of Pyridoxal-P
and NaCN
Lysyl residues have been implicated in substrate and coenzyme binding and in catalysis for a number of enzymes (15-17). Pyridoxal-P is useful for chemical modification of lysyl and amino terminal residues (15, 16, 18). To determine whether free amino groups were essential for anthranilate synthase activity the enzyme was incubated with pyridoxal-P under different conditions. The data in Fig. 1 show a time-dependent inactivation of anthranilate synthase. Inactivation required the simultaneous presence of pyridoxal-P and NaCN. Glutamine-dependent anthranilate synthase was specifically inactivated. The NH3-dependent activity was not affected. Chorismate, a substrate, stimulated the rate of amidotransferase inactivation. These results implicate an essential residue involved in the binding or utilization of glutamine by the enzyme. The rate of inactivation was dependent upon the concentrations of both NaCN and pyridoxal-P. At 10 mM NaCN in the presence of 0.5 mM chorismate and over the pyridoxal-P concentration range of 0.5 to 10 mM a second-order rate constant of 118 Mpl mini’ was determined. A second order rate constant of 6.6 Me’ mini’ was determined with 5 mM pyridoxal-P, 0.5 mM chorismate, and 1 to 10 mM NaCN. These results are consistent with formation of a lysyl-pyridoxal-P Schiff base that is stabilized by NaCN and is enzymatically inactive. The chemically modified enzyme was not reactivated by dialysis suggesting stable covalent attachment of pyridoxal-P 4 J. Tso, unpublished
data.
ANTHRANILATE
SYNTHASE
MODIFICATION
(Table I). As shown in Table I, the enzyme was reactivated by dithiothreitol. Inactivation
by Pyridoxal-P
BY
123
PYRIDOXAL-P
TABLE
I
EFFECT OF DIALYSISON~NACTIVATED ANTHRANILATE~YNTHASE'
and NaBH,
Anthranilate synthase inactivation was also obtained by pyridoxal-P and NaBH4. This chemical modification differed significantly from that shown in Fig. 1 using pyridoxal-P and NaCN. A 16-fold higher pyridoxal-P concentration gave only 70% inactivation compared to the greater than 95% inactivation obtained with pyridoxalP and NaCN. Furthermore, the inactivation was not specific for the glutaminedependent activity. Inactivation of NHBdependent anthranilate synthase was approximately 75% that of the glutaminedependent activity. Control experiments verified that NaBH4 by itself was not inhibitory. Inactivation of anthranilate synthase by pyridoxal-P likely results from Schiff base formation with protein amino groups. Direct evidence for lysyl modification was
Pyridoxal-P -
Activity CA)
Dithiothreitol in dialysis -
100 100
+
+
4
-
+
4
+
Activity dialysis
after (%)
9s 102 6 IS
’ Anthranilate synthase (8 pM) was inactivated with 5 mM pyridoxal-P and 10 rn~ N&N. In a control reaction, the pyridoxal-P was omitted. The treated enzymes were dialyzed against 50 mM potassium phosphate pH 7.4/0.1 mM EDTA. Where noted 0.2 mM dithiothreitol was included in dialysis.
obtained using NaBH, to reduce enzyme treated with 80 mM pyridoxal-P. Amino acid analysis indicated loss of approximately 12 of 30 iysyl residues in the AS 1. AS II protomer from an enzyme that was 70% inactivated. Spectral Properties of Enzyme Modified with Pyridoxal-P and NaCN The spectrum of anthranilate synthase modified with pyridoxal-P and NaCN had an absorbance maximum near 325 nm (Fig. 2). The position of maximum absorbance is virtually identical with that observed for lysyl Schiff base adducts reduced with NaBH4 (15, 16, 18).
100 SO 60
Stoichiometry of Inactivation
1
0 FIG. pyridoxal-P
I
5
1. Inactivation plus NaCN.
I
I
IO TIME
of
15 (mtnutes)
anthranilate Enzyme
20
(8
synthase PM) was
by incu-
bated in 50 mM potassium phosphate pH 7.4 with additions indicated: (0) 10 mM NaCN, (0) 5 mM pyridoxal-P, (0) 10 mM NaCN and 5 mM pyridoxal-P, (m) 10 mM NaCN, 5 mM pyridoxal-P and 0.5 mM chorismate. Samples of 0.01 ml were removed and assayed for glutamine-dependent anthranilate synthase activity.
The stoichiometry for modification of anthranilate synthase with pyridoxal-P was determined using [14C]NaCN. Approximately 0.5 eq of 14CN was incorporated into the AS 1. AS II protomer in the absence of pyridoxal-P (Table II). Little or no inactivation was associated with this incorporation. With pyridoxal-P present the enzyme was completely inactivated and 1 additional eq of 14CN per enzyme protomer was incorporated. Distinction between the Essential Cysteinyl and Lysyl Residues in Glutamine Amide Transfer Glutamine amide transfer function requires at least three steps: (a) glutamine
124
BOWER
AND
FIG. 2. Spectra of native and pyridoxal5’phosphate modified anthranilate synthase. Anthranilate synthase was incubated with 10 mM NaCN (- - -1 or 10 mM NaCN and 5 mM pyridoxal-P (p) for 15 min. Excess reagents were removed by dialysis against 10 mM potassium Hepes pH 7.5 containing 0.1 mM EDTA. Enzyme concentrations were adjusted to 667 pM and spectra were determined.
binding, (b) formation of the covalent glutaminyl intermediate with the active site cysteinyl residue, and (c) amide transfer to chorismate and release of glutamate. Modification of enzyme with pyridoxal-P and NaCN could disrupt any of these steps. Affinity labeling of anthranilate synthase by the glutamine analog DON requires only the initial two steps: noncovalent binding to the substrate site followed by alkylation of the active site cysteinyl residue (5, 9, 19). The data in Table III show the expected inactivation of anthranilate synthase by DON. The stoichiometry of affinity labeling was 0.76 eq DON incorporated per protomer for 80% inactivation. Initial inactivation by pyridoxalP plus NaCN prevented affinity labeling by DON. Thus lysyl modification prevents either glutamine binding or formation of the covalent glutaminyl intermediate. Iodoacetamide has been used for selective chemical modification of the active
ZALKIN
site cysteinyl residue in a second-order reaction that is independent of initial binding to the glutamine site (5, 7). The data in Table IV show inactivation of glutamine-dependent activity by iodoacetamide. The stoichiometry of alkylation was 0.93 eq carboxamidomethyl incorporated per protomer for 90% inactivation. Initial lysyl modification reduced alkylation to 0.35 eq per protomer. It was not determined whether this latter carboxamidomethylation was specific for active site Cys-83 (7) but it is apparent that prior lysyl modification decreases the reactivity of the active site cysteinyl residue. The data in Table V show that lysyl modification by pyridoxal-P and NaCN is independent of the active site cysteinyl residue. In the control experiment 1.27 eq of 14CN was incorporated per protomer while incorporation of 1.40 eq of 14CN per protomer was obtained for enzyme previously alkylated with iodoacetamide. These data are uncorrected for the approximately 0.5 eq per protomer of 14CN that is nonspecifically incorporated. DISCUSSION
The main conclusion of this study is that the glutamine amidotransferase function of anthranilate synthase is specifically inactivated by chemical modification of a single amino acid residue by pyridoxal-P plus NaCN. The essential residue is disTABLE
Reaction conditions
Inactivation halftime (min)
Complete Chorismate -Pyridoxal-P +Glutamine
1.5 2.6 62 3.0
II
‘%N
per protomer (es) 1.3 1.4 0.5 1.1
a The reaction mixture included 10 pM enzyme, 0.5 mM chorismate, 10 mM [“C]NaCN, and 0.5 mM pyridoxal-P. Incubation was for 20 min. Glutamine when present was 10 mM.
ANTHRANILATE
SYNTHASE
MODIFICATION
tinct from the AS II active site cysteinyl residue and is likely a lysyl residue with unique properties. Pyridoxal-P is a highly specific reagent for amino group modification (15, 16, 18). In all cases the initial step in lysyl modification by pyridoxal-P is Schiff base formation. The initial Schiff base may vary in stability but upon dilution is subject to hydrolysis with concomitant regeneration of enzyme activity. The anthranilate synthase pyridoxal-P Schiff base must be extremely labile because the treated enzyme is essentially fully active when diluted and assayed. With some enzymes the initial lysyl-pyridoxal-P Schiff base undergoes a secondary reaction with a reactive nucleophile such as a sulfhydryl to form an X-azolidine (20). X-axolidine adducts are irreversible in the absence of reduction (20). Since the anthranilate synthase pyridoxal-P adduct is extremely labile, X-azolidine formation is excluded. This conclusion is further supported by the finding that alkylation of the AS II active site cysteinyl residue did not reduce binding of pyridoxal-P (Table V). Similar conclusions were drawn by Paech and Tolbert using ribulose-1,5-bisphosphate carboxylase/oxygenase (21). TABLE
III
PYRIDOXAL 5’-PHOSPHATE MODIFICATION BLOCKS DON INCORPORATION’ Pyridoxal-P modification Initial concentration bM) 0 0.5
[6-%]DON incorporation
Activity (%)
Activity (%)
98 1
20 <1
Incorporation per protomer (es) 0.76 0
“Anthranilate synthase (9.4 PM) was incubated without or with 0.5 mM pyridoxal-P in the presence of 0.5 mM chorismate and 10 mM NaCN. After 20 min, excess reagents were removed by dialysis against 50 mM potassium Hepes pH 7.5/0.1 mM EDTA and glutamine-dependent activity was determined. Enzyme samples were then treated with 0.02 mM [6‘%]DON for 20 min and excess reagent was removed by dialysis as above. Incorporation was determined from radioactivity and amino acid analysis.
BY
TABLE PYRIDOXAL-5’-PHOSPHATE I~DOACETAMIDE Pyridoxal-P modification Initial concentration bM) 0 0.5
125
PYRIDOXAL-P IV MODIFICATION INCORPORATION”
REDUCES
[ l-‘4C]Iodoacetamide incorporation
Activity (%)
Activity (%)
104 <1
10 <1
Incorporation per protomer (es) 0.93 0.35
” Anthranilate synthase was modified with pyridoxal-P as described in footnote a to Table III. After 10 min the inactivation mixtures were made 2.5 mM in [1-“Cliodoacetamide. Incubation was for 20 min and then samples were freed of excess reagents by centrifugation gel filtration and incorporation was determined.
The anthranilate synthase active site Schiff base adduct is uniquely stabilized by NaCN but not by NaBH4. The evidence suggests that an essential amino group, a lysyl, or amino terminal residue was modified by pyridoxal-P. First, the adduct stabilized by NaCN exhibits an absorption spectrum similar to that of a reduced Schiff base. Second, we are unaware of alternative amino acid side chains that undergo stable modification by pyridoxalP. The fact that neither NaCN nor pyridoxal-P alone inactivates anthranilate synthase but the two reagents together cause inactivation strongly supports a mechanism of inactivation that involves the addition
of HCN
to the >C=N<
of
a Schiff base. Although addition of HCN to the pyridoxal-P-lysyl Schiff base has apparently not been reported, such additions are well known in aldolase and transketolase enzyme-substrate intermediates (22-25). In most cases these aminonitriles readily dissociate upon dilution or dialysis (23, 25). In the only reported case of a stable HCN-azomethine adduct, the addition product was also stable to dialysis against mercaptoethanol (24). The HCN addition product of pyridoxal-P-modified anthranilate synthase was stable to dialysis but
126
BOWER
TABLE
Iodoacetamide modification Initial concentration (I-m) 0 2.5
AND
V
Pyridoxal-P-dependent “CN incorporation
Activity
Activity
(%)
(%)
97 <1
10
Incorporation per protomer (eq) 1.27 1.40
’ Anthranilate synthase (9.6 PM) was treated with iodoacetamide for 30 min under conditions described in footnote (I to Table III. Following carboxamidomethylation 0.5 mM pyridoxal-P and 10 mM ['%]NaCN were added to the inactivation mixtures. After 10 min samples were freed of excess reagents by centrifugation gel filtration and incorporation was determined.
was reversed by dithiothreitol and thus exhibits unique stability. Although anthranilate synthase was irreversibly inactivated by pyridoxal-P followed by NaBH, reduction, this modification reaction clearly differed from that employing NaCN. First, a greater than 20fold higher concentration of pyridoxal-P was required for comparable inactivation. Second, the higher pyridoxal-P concentration employed with NaBH4 reduction lead to increased lysyl modification: 12 lysyl residues modified per protomer (at 70% inactivation) after NaBH4 reduction compared to 1 lysyl modified per protomer (at 99% inactivation) using NaCN addition. Finally, both the NH3- and glutamine-dependent activities were inactivated by pyridoxal-P and NaBH4 in contrast to specific inactivation of glutamine-dependent anthranilate synthase by pyridoxal-P and NaCN. It appears that enhanced selectively for modification of the glutamine amidotransferase active site lysyl residue when using NaCN to stabilize the putative Schiff base results from the lower concentration of pyridoxal-P relative to that required with NaBH4 reduction. Selective inactivation of glutamine-dependent anthranilate synthase indicates modification of a residue involved in glu-
ZALKIN
tamine binding, formation of the covalent glutaminyl-enzyme intermediate, or amide transfer to chorismate. The data in Tables III and IV provide evidence that the essential lysyl residue enhances the reactivity of the active site cysteinyl residue. One possibility is that the lysyl residue functions as a general acid-base to promote ionization of the cysteinyl residue. Alternatively, the very proximity of a positively charged lysyl residue, especially in a hydrophobic environment, would depress the pK, for the active site cysteinyl residue. Reactivity of the AS II active site cysteinyl residue for formation of the acyl-enzyme intermediate (19) and modification by the glutamine affinity analog, DON (9) is enhanced by binding of chorismate to AS I. The rate of lysyl modification is also enhanced by chorismate, providing further evidence for interaction of these two residues involved in the glutamine amide transfer function of anthranilate synthase. Anthranilate synthase glutamine amide transfer function is dependent upon AS II. Amino acid sequences have been determined for the AS II subunit of anthranilate synthase from five organisms including 5’. marcescens (5-7). Alignment of these protein chains indicates 76 invarient residues out of 192 amino acids. In addition to conservation of active site Cys-83, Lys-107 (using S. marcescew numbering) is conserved in all of the AS II proteins and is situated in a block of highly conserved residues. S. marcescens Lys-192 is conserved in four of the AS II proteins but is replaced by Arg-196 in the Pseudomonas putida enzyme. S. marcescens lysines 107 and 192 are thus candidates for the essential residue modified by pyridoxal-P and NaCN. ACKNOWLEDGMENTS We thank Dr. John S. Holcenberg for the gift of N-trifluoracetyl-L-glutamic-y-acid chloride-a-methyl ester and for his detailed notes on synthesis of DON. We are grateful to J. Yun Tso for preparing the anthranilate synthase. REFERENCES
1. ZALKIN, H. (1973) in The Enzymes
of Glutamine Metabolism (Prusiner, S., and Stadtman, E., eds.), pp. 523-543, Academic Press, New York.
ANTHRANILATE
SYNTHASE
MODIFICATION
2. ZALKIN, H. (1980) in Multifunctional Proteins (Bisswanger, H., and Schmincke-Ott, E., eds.), pp. 1233149, John Wiley, New York. 3. Tso, J., BOWER, S., AND ZALKIN, H. (1980) J. Biol. Chem. 255, 6734-6738. 4. NICHOLS, B., VAN CLEEMPUT, M., AND YANOFSKY, C. (1981) J. Mol. Biol. 146, 45-54. 5. KAWAMURA, M., KEIM, P., GOTO, Yo, ZALKIN, H., AND HEINRIKSON, R. (1978) J. Biol. Chem.
BY
127
PYRIDOXAL-P
14. PENEFSKY, H. S. (1977) J. Biol. Chem. 252, 2891-2899. 15. OGAWA, H., AND FUJIOKA, M. (1980) J. Biol. Chem. 255, 7420-7425. 16. NEUJAHR, H., AND KJELLEN, K. (1980) Biochem-
istry 19,4967-4972. 17. MUHLRAD, A., AND TAKASHI, istry 20,6749-6754.
R. (1981)
Biochem-
6. NICHOLS,
18. GLAZER, A., DELANGE, R., AND SIGMAN, D. (1975) Chemical Modification of Proteins, pp. 131134, North-Holland/American Elsevier, New York.
7.
19. GOTO, Y., ZALKIN, H., KEIM, P., AND HEINRIKSON, R. (1976) J. Biol. Chem. 251, 941-949.
253,4659-4668.
8. 9. 10. 11.
B., MIOZZARI, G., VAN CLEEMPUT, M., BENNETT, G., AND YANOFSKY, C. (1980) J. Mol. Biol. 142, 503-518. Tso, J., HERMODSON, M., AND ZALKIN, H. (1980) J. Biol. Chem. 255, 1451-1457. Tso, J., AND ZALKIN, H. (1981) J. Biol. Chem. 256, 9901-9908. NAGANO, H., ZALKIN, H., AND HENDERSON, E. (1970) J. Biol. Chem. 245, 3810-3820. Tso, J., HERMODSON, M. A., AND ZALKIN, H. (1982) J. Biol. Chem. 257, 3532-3536. HARTMAN, S. C. (1963) J. Biol. Chem. 238,3036-
3047. 12. HENDERSON, E., NAGANO, H., ZALKIN, H., AND HWANG, L. (1970) J. Biol. Chem. 245, 14161423. 13. ZALKIN, H., AND KLING, D. (1968) Biochemistry
7, 3566-3573.
20. BLEILE, (1976)
D., JAMESON, J. Biol. C&m.
J.,
AND
HARRISON,
21. PAECH, C., AND TOLBERT, Chem. 253, 7864-7873.
N.
(1978)
22. BRAND, K., AND HORECKER, B. Biochem. Biophys. 123, 312-316. 23. AUTOR, A., AND FRIDOVICH, Chem. 245, 5214-5222.
241.4290-4292.
J.
(1968)
I. (1970)
24. HANSEN, B., LANE, R., AND DEKKER, J. Biol. Chem. 249, 4891-4896. 25. CASH, D., AND WILSON,
J.
251, 6304-6307.
I. (1966)
Biol. A&L
J. Biol. E. (1974)
J. Biol.
Chem.
Vol.
219,
OF BIOCHEMISTRY
No.
AND
1, November,
pp.
Modification
BIOPHYSICS 121-127, 1982
of Serratia marcescens Anthranilate with Pyridoxal S-Phosphate’ STANLEY
Department
BOWER
of Biochemistry,
AND
Purdue
HOWARD
Uniuersity,
Received
June
West
Synthase
ZALKIN2 Lafayette,
Indiana
47907
10, 1982
The glutamine-dependent activity of Serrutia marcescens anthranilate synthase was inactivated by pyridoxal 5’-phosphate and sodium cyanide. The reaction was specific in that the ammonia-dependent activity of the enzyme was unaffected. The inactivation was stable to dilution or dialysis hut was reversed by dithiothreitol. The enzyme contains dissimilar subunits designated anthranilate synthase components I (AS I) and II (AS II). Incorporation of [14C]NaCN demonstrates that modification was limited to one to two residues per AS 1. AS II protomer. An active site cysteine is involved in the glutamine-dependent activity. Modification by pyridoxal 5’-phosphate and NaCN blocked affinity labeling of the active site cysteine by the glutamine analog 6-diazo-5-0x0-b norleucine and reduced alkylation of the active site cysteine by iodoacetamide. These results suggest modification is at the glutamine active site. Initial modification by iodoacetamide did not prevent pyridoxal 5’-phosphate-dependent incorporation of 14CN showing that the pyridoxal 5’-phosphate modification did not involve the essential cysteinyl residue. These results suggest that modification of a lysyl residue in the glutamine active site of anthranilate synthase reduces the reactivity of the essential cysteinyl residue resulting in the loss of the amidotransferase activity.
Anthranilate synthase catalyzes the first reaction of tryptophan biosynthesis. The enzyme from enteric bacteria is an oligomerit protein containing dissimilar suhunits designated AS I and AS II.3 AS I catalyzes the reaction shown in Eq. [l]:
AS II provides glutamine amidotransferase function for the oligomeric enzyme complex (Eq. [Z]). Chorismate Anthranilate
M$+
Chorismate
+ glutamine + pyruvate
+ glutamate
+ NH3 Anthranilate
Mg*+
-
+ pyruvate
[2]
Anthranilate synthase is one of the best understood of the many glutamine amidotransferases in prokaryotic and eukaryotic cells (1, 2) and has served as a model for understanding more complex enzymes of this type (3). The amino acid sequences of the AS I subunit from Escherichia coli and Salmonella typhimurium (4) have been deduced from the DNA sequences. Amino acid sequences of AS IT subunits from several bacteria have been obtained by protein and DNA sequencing (5-7). Amino acid residues have been identified which are
[l]
’ This work was supported hy Public Health Service Grant GM 24658 from the National Institute of General Medical Sciences. This is Journal Paper Number 9052 from the Purdue Agricultural Experiment Station. ‘Author to whom correspondence should be addressed. ’ Abbreviations used: AS I, anthranilate synthase component I; AS II, anthranilate synthase component II; pyridoxal-P, pyridoxal %phosphate; DON, 6-diazo5-oxo-1,.norleucine; Hepes, N-2-hydroxyethylpiperazinc-l\i’-2.ethane sulfonic acid. 121
000%9861/82/130121-07$02.00/O Copyright Q 1982 by Academic All
rights
of reproduction
Press, Inc.
in any form
reserved.
122
BOWER
AND
essential to the function of each subunit (7, 8). Single arginyl, histidyl, and cysteinyl residues in AS I were found to be essential for the NH,-dependent reaction (8). The roles of these residues are not clearly understood but they appear to be involved with binding of chorismate and its conversion to anthranilate plus pyruvate. Glutamine amidotransferase function requires an AS II active site cysteinyl residue (7). The role of this active site cysteinyl residue in activation of glutamine and amide transfer was first worked out for anthranilate synthase (9) and appears to be similar for other glutamine amidotransferases (10). In this report we provide the first evidence for a second residue essential for the glutamine amide transfer function. This amino acid was modified by treatment of anthranilate synthase with pyridoxal-P. The essential residue exhibits properties of an unique lysyl residue. MATERIALS
AND
METHODS
Materials. Anthranilate synthase was purified from S. marcescens (ATCC 27143) by J. Yun Tso (7). [6‘%]DON was prepared by the method of Hartman (11) from N-trifluoroacetyl-L-glutamic-y-acid chloride-a-methyl ester which was a generous gift from Dr. John S. Holcenberg, Medical College of Wisconsin. Iodo[l-%]acetamide and [“C]NaCN were from New England Nuclear. Most other chemicals were commercial products of the highest purity available and were used without further purification. Methods. Anthranilate synthase was assayed as previously described (12, 13). Inactivations were performed in 50 mM potassium phosphate pH 7.4 containing 0.1 mM EDTA. The reactions were conducted at room temperature and were initiated by the addition of pyridoxal-P, DON, or iodoacetamide. Aliquots of the reaction mixture were diluted loo-fold into the standard assay mixture. In some cases aliquots of up to 100 ~1 were rapidly desalted by centrifugation gel filtration (14). Excess reagents were removed from preparative scale modifications by dialysis. When inactivation was by pyridoxal-P and NaBH,, anthranilate synthase was incubated in 0.25 ml of 50 mM potassium phosphate pH 7.4, 0.1 mM EDTA and pyridoxal-P. After 20 min 20 ~1 of n-octanol was added followed by 0.25 ml of 0.1 M NaBH,. After a 5-min incubation at 4°C samples were dialyzed against 50 mM potassium phosphate pH 7.4, 0.1 mM EDTA, and 0.2 mM dithiothreitol. Activity was determined by the standard assay. Protein con-
ZALKIN centration and the number of lysyl residues modified were determined by amino acid analysis. Second-order rate constants for enzyme inactivation were determined from the slope of plots of pseudo-first-order rate constants versus reagent concentration. Data were fit by linear regression. Anthranilate synthase concentrations were routinely determined by absorbance at 278 nm using an extinction coehicient, &” = 0.658,’ which was confirmed by amino acid analysis. Amino acid analyses were performed with a Durrum D 500 amino acid analyzer. RESULTS
Inactivation
of Pyridoxal-P
and NaCN
Lysyl residues have been implicated in substrate and coenzyme binding and in catalysis for a number of enzymes (15-17). Pyridoxal-P is useful for chemical modification of lysyl and amino terminal residues (15, 16, 18). To determine whether free amino groups were essential for anthranilate synthase activity the enzyme was incubated with pyridoxal-P under different conditions. The data in Fig. 1 show a time-dependent inactivation of anthranilate synthase. Inactivation required the simultaneous presence of pyridoxal-P and NaCN. Glutamine-dependent anthranilate synthase was specifically inactivated. The NH3-dependent activity was not affected. Chorismate, a substrate, stimulated the rate of amidotransferase inactivation. These results implicate an essential residue involved in the binding or utilization of glutamine by the enzyme. The rate of inactivation was dependent upon the concentrations of both NaCN and pyridoxal-P. At 10 mM NaCN in the presence of 0.5 mM chorismate and over the pyridoxal-P concentration range of 0.5 to 10 mM a second-order rate constant of 118 Mpl mini’ was determined. A second order rate constant of 6.6 Me’ mini’ was determined with 5 mM pyridoxal-P, 0.5 mM chorismate, and 1 to 10 mM NaCN. These results are consistent with formation of a lysyl-pyridoxal-P Schiff base that is stabilized by NaCN and is enzymatically inactive. The chemically modified enzyme was not reactivated by dialysis suggesting stable covalent attachment of pyridoxal-P 4 J. Tso, unpublished
data.
ANTHRANILATE
SYNTHASE
MODIFICATION
(Table I). As shown in Table I, the enzyme was reactivated by dithiothreitol. Inactivation
by Pyridoxal-P
BY
123
PYRIDOXAL-P
TABLE
I
EFFECT OF DIALYSISON~NACTIVATED ANTHRANILATE~YNTHASE'
and NaBH,
Anthranilate synthase inactivation was also obtained by pyridoxal-P and NaBH4. This chemical modification differed significantly from that shown in Fig. 1 using pyridoxal-P and NaCN. A 16-fold higher pyridoxal-P concentration gave only 70% inactivation compared to the greater than 95% inactivation obtained with pyridoxalP and NaCN. Furthermore, the inactivation was not specific for the glutaminedependent activity. Inactivation of NHBdependent anthranilate synthase was approximately 75% that of the glutaminedependent activity. Control experiments verified that NaBH4 by itself was not inhibitory. Inactivation of anthranilate synthase by pyridoxal-P likely results from Schiff base formation with protein amino groups. Direct evidence for lysyl modification was
Pyridoxal-P -
Activity CA)
Dithiothreitol in dialysis -
100 100
+
+
4
-
+
4
+
Activity dialysis
after (%)
9s 102 6 IS
’ Anthranilate synthase (8 pM) was inactivated with 5 mM pyridoxal-P and 10 rn~ N&N. In a control reaction, the pyridoxal-P was omitted. The treated enzymes were dialyzed against 50 mM potassium phosphate pH 7.4/0.1 mM EDTA. Where noted 0.2 mM dithiothreitol was included in dialysis.
obtained using NaBH, to reduce enzyme treated with 80 mM pyridoxal-P. Amino acid analysis indicated loss of approximately 12 of 30 iysyl residues in the AS 1. AS II protomer from an enzyme that was 70% inactivated. Spectral Properties of Enzyme Modified with Pyridoxal-P and NaCN The spectrum of anthranilate synthase modified with pyridoxal-P and NaCN had an absorbance maximum near 325 nm (Fig. 2). The position of maximum absorbance is virtually identical with that observed for lysyl Schiff base adducts reduced with NaBH4 (15, 16, 18).
100 SO 60
Stoichiometry of Inactivation
1
0 FIG. pyridoxal-P
I
5
1. Inactivation plus NaCN.
I
I
IO TIME
of
15 (mtnutes)
anthranilate Enzyme
20
(8
synthase PM) was
by incu-
bated in 50 mM potassium phosphate pH 7.4 with additions indicated: (0) 10 mM NaCN, (0) 5 mM pyridoxal-P, (0) 10 mM NaCN and 5 mM pyridoxal-P, (m) 10 mM NaCN, 5 mM pyridoxal-P and 0.5 mM chorismate. Samples of 0.01 ml were removed and assayed for glutamine-dependent anthranilate synthase activity.
The stoichiometry for modification of anthranilate synthase with pyridoxal-P was determined using [14C]NaCN. Approximately 0.5 eq of 14CN was incorporated into the AS 1. AS II protomer in the absence of pyridoxal-P (Table II). Little or no inactivation was associated with this incorporation. With pyridoxal-P present the enzyme was completely inactivated and 1 additional eq of 14CN per enzyme protomer was incorporated. Distinction between the Essential Cysteinyl and Lysyl Residues in Glutamine Amide Transfer Glutamine amide transfer function requires at least three steps: (a) glutamine
124
BOWER
AND
FIG. 2. Spectra of native and pyridoxal5’phosphate modified anthranilate synthase. Anthranilate synthase was incubated with 10 mM NaCN (- - -1 or 10 mM NaCN and 5 mM pyridoxal-P (p) for 15 min. Excess reagents were removed by dialysis against 10 mM potassium Hepes pH 7.5 containing 0.1 mM EDTA. Enzyme concentrations were adjusted to 667 pM and spectra were determined.
binding, (b) formation of the covalent glutaminyl intermediate with the active site cysteinyl residue, and (c) amide transfer to chorismate and release of glutamate. Modification of enzyme with pyridoxal-P and NaCN could disrupt any of these steps. Affinity labeling of anthranilate synthase by the glutamine analog DON requires only the initial two steps: noncovalent binding to the substrate site followed by alkylation of the active site cysteinyl residue (5, 9, 19). The data in Table III show the expected inactivation of anthranilate synthase by DON. The stoichiometry of affinity labeling was 0.76 eq DON incorporated per protomer for 80% inactivation. Initial inactivation by pyridoxalP plus NaCN prevented affinity labeling by DON. Thus lysyl modification prevents either glutamine binding or formation of the covalent glutaminyl intermediate. Iodoacetamide has been used for selective chemical modification of the active
ZALKIN
site cysteinyl residue in a second-order reaction that is independent of initial binding to the glutamine site (5, 7). The data in Table IV show inactivation of glutamine-dependent activity by iodoacetamide. The stoichiometry of alkylation was 0.93 eq carboxamidomethyl incorporated per protomer for 90% inactivation. Initial lysyl modification reduced alkylation to 0.35 eq per protomer. It was not determined whether this latter carboxamidomethylation was specific for active site Cys-83 (7) but it is apparent that prior lysyl modification decreases the reactivity of the active site cysteinyl residue. The data in Table V show that lysyl modification by pyridoxal-P and NaCN is independent of the active site cysteinyl residue. In the control experiment 1.27 eq of 14CN was incorporated per protomer while incorporation of 1.40 eq of 14CN per protomer was obtained for enzyme previously alkylated with iodoacetamide. These data are uncorrected for the approximately 0.5 eq per protomer of 14CN that is nonspecifically incorporated. DISCUSSION
The main conclusion of this study is that the glutamine amidotransferase function of anthranilate synthase is specifically inactivated by chemical modification of a single amino acid residue by pyridoxal-P plus NaCN. The essential residue is disTABLE
Reaction conditions
Inactivation halftime (min)
Complete Chorismate -Pyridoxal-P +Glutamine
1.5 2.6 62 3.0
II
‘%N
per protomer (es) 1.3 1.4 0.5 1.1
a The reaction mixture included 10 pM enzyme, 0.5 mM chorismate, 10 mM [“C]NaCN, and 0.5 mM pyridoxal-P. Incubation was for 20 min. Glutamine when present was 10 mM.
ANTHRANILATE
SYNTHASE
MODIFICATION
tinct from the AS II active site cysteinyl residue and is likely a lysyl residue with unique properties. Pyridoxal-P is a highly specific reagent for amino group modification (15, 16, 18). In all cases the initial step in lysyl modification by pyridoxal-P is Schiff base formation. The initial Schiff base may vary in stability but upon dilution is subject to hydrolysis with concomitant regeneration of enzyme activity. The anthranilate synthase pyridoxal-P Schiff base must be extremely labile because the treated enzyme is essentially fully active when diluted and assayed. With some enzymes the initial lysyl-pyridoxal-P Schiff base undergoes a secondary reaction with a reactive nucleophile such as a sulfhydryl to form an X-azolidine (20). X-axolidine adducts are irreversible in the absence of reduction (20). Since the anthranilate synthase pyridoxal-P adduct is extremely labile, X-azolidine formation is excluded. This conclusion is further supported by the finding that alkylation of the AS II active site cysteinyl residue did not reduce binding of pyridoxal-P (Table V). Similar conclusions were drawn by Paech and Tolbert using ribulose-1,5-bisphosphate carboxylase/oxygenase (21). TABLE
III
PYRIDOXAL 5’-PHOSPHATE MODIFICATION BLOCKS DON INCORPORATION’ Pyridoxal-P modification Initial concentration bM) 0 0.5
[6-%]DON incorporation
Activity (%)
Activity (%)
98 1
20 <1
Incorporation per protomer (es) 0.76 0
“Anthranilate synthase (9.4 PM) was incubated without or with 0.5 mM pyridoxal-P in the presence of 0.5 mM chorismate and 10 mM NaCN. After 20 min, excess reagents were removed by dialysis against 50 mM potassium Hepes pH 7.5/0.1 mM EDTA and glutamine-dependent activity was determined. Enzyme samples were then treated with 0.02 mM [6‘%]DON for 20 min and excess reagent was removed by dialysis as above. Incorporation was determined from radioactivity and amino acid analysis.
BY
TABLE PYRIDOXAL-5’-PHOSPHATE I~DOACETAMIDE Pyridoxal-P modification Initial concentration bM) 0 0.5
125
PYRIDOXAL-P IV MODIFICATION INCORPORATION”
REDUCES
[ l-‘4C]Iodoacetamide incorporation
Activity (%)
Activity (%)
104 <1
10 <1
Incorporation per protomer (es) 0.93 0.35
” Anthranilate synthase was modified with pyridoxal-P as described in footnote a to Table III. After 10 min the inactivation mixtures were made 2.5 mM in [1-“Cliodoacetamide. Incubation was for 20 min and then samples were freed of excess reagents by centrifugation gel filtration and incorporation was determined.
The anthranilate synthase active site Schiff base adduct is uniquely stabilized by NaCN but not by NaBH4. The evidence suggests that an essential amino group, a lysyl, or amino terminal residue was modified by pyridoxal-P. First, the adduct stabilized by NaCN exhibits an absorption spectrum similar to that of a reduced Schiff base. Second, we are unaware of alternative amino acid side chains that undergo stable modification by pyridoxalP. The fact that neither NaCN nor pyridoxal-P alone inactivates anthranilate synthase but the two reagents together cause inactivation strongly supports a mechanism of inactivation that involves the addition
of HCN
to the >C=N<
of
a Schiff base. Although addition of HCN to the pyridoxal-P-lysyl Schiff base has apparently not been reported, such additions are well known in aldolase and transketolase enzyme-substrate intermediates (22-25). In most cases these aminonitriles readily dissociate upon dilution or dialysis (23, 25). In the only reported case of a stable HCN-azomethine adduct, the addition product was also stable to dialysis against mercaptoethanol (24). The HCN addition product of pyridoxal-P-modified anthranilate synthase was stable to dialysis but
126
BOWER
TABLE
Iodoacetamide modification Initial concentration (I-m) 0 2.5
AND
V
Pyridoxal-P-dependent “CN incorporation
Activity
Activity
(%)
(%)
97 <1
10
Incorporation per protomer (eq) 1.27 1.40
’ Anthranilate synthase (9.6 PM) was treated with iodoacetamide for 30 min under conditions described in footnote (I to Table III. Following carboxamidomethylation 0.5 mM pyridoxal-P and 10 mM ['%]NaCN were added to the inactivation mixtures. After 10 min samples were freed of excess reagents by centrifugation gel filtration and incorporation was determined.
was reversed by dithiothreitol and thus exhibits unique stability. Although anthranilate synthase was irreversibly inactivated by pyridoxal-P followed by NaBH, reduction, this modification reaction clearly differed from that employing NaCN. First, a greater than 20fold higher concentration of pyridoxal-P was required for comparable inactivation. Second, the higher pyridoxal-P concentration employed with NaBH4 reduction lead to increased lysyl modification: 12 lysyl residues modified per protomer (at 70% inactivation) after NaBH4 reduction compared to 1 lysyl modified per protomer (at 99% inactivation) using NaCN addition. Finally, both the NH3- and glutamine-dependent activities were inactivated by pyridoxal-P and NaBH4 in contrast to specific inactivation of glutamine-dependent anthranilate synthase by pyridoxal-P and NaCN. It appears that enhanced selectively for modification of the glutamine amidotransferase active site lysyl residue when using NaCN to stabilize the putative Schiff base results from the lower concentration of pyridoxal-P relative to that required with NaBH4 reduction. Selective inactivation of glutamine-dependent anthranilate synthase indicates modification of a residue involved in glu-
ZALKIN
tamine binding, formation of the covalent glutaminyl-enzyme intermediate, or amide transfer to chorismate. The data in Tables III and IV provide evidence that the essential lysyl residue enhances the reactivity of the active site cysteinyl residue. One possibility is that the lysyl residue functions as a general acid-base to promote ionization of the cysteinyl residue. Alternatively, the very proximity of a positively charged lysyl residue, especially in a hydrophobic environment, would depress the pK, for the active site cysteinyl residue. Reactivity of the AS II active site cysteinyl residue for formation of the acyl-enzyme intermediate (19) and modification by the glutamine affinity analog, DON (9) is enhanced by binding of chorismate to AS I. The rate of lysyl modification is also enhanced by chorismate, providing further evidence for interaction of these two residues involved in the glutamine amide transfer function of anthranilate synthase. Anthranilate synthase glutamine amide transfer function is dependent upon AS II. Amino acid sequences have been determined for the AS II subunit of anthranilate synthase from five organisms including 5’. marcescens (5-7). Alignment of these protein chains indicates 76 invarient residues out of 192 amino acids. In addition to conservation of active site Cys-83, Lys-107 (using S. marcescew numbering) is conserved in all of the AS II proteins and is situated in a block of highly conserved residues. S. marcescens Lys-192 is conserved in four of the AS II proteins but is replaced by Arg-196 in the Pseudomonas putida enzyme. S. marcescens lysines 107 and 192 are thus candidates for the essential residue modified by pyridoxal-P and NaCN. ACKNOWLEDGMENTS We thank Dr. John S. Holcenberg for the gift of N-trifluoracetyl-L-glutamic-y-acid chloride-a-methyl ester and for his detailed notes on synthesis of DON. We are grateful to J. Yun Tso for preparing the anthranilate synthase. REFERENCES
1. ZALKIN, H. (1973) in The Enzymes
of Glutamine Metabolism (Prusiner, S., and Stadtman, E., eds.), pp. 523-543, Academic Press, New York.
ANTHRANILATE
SYNTHASE
MODIFICATION
2. ZALKIN, H. (1980) in Multifunctional Proteins (Bisswanger, H., and Schmincke-Ott, E., eds.), pp. 1233149, John Wiley, New York. 3. Tso, J., BOWER, S., AND ZALKIN, H. (1980) J. Biol. Chem. 255, 6734-6738. 4. NICHOLS, B., VAN CLEEMPUT, M., AND YANOFSKY, C. (1981) J. Mol. Biol. 146, 45-54. 5. KAWAMURA, M., KEIM, P., GOTO, Yo, ZALKIN, H., AND HEINRIKSON, R. (1978) J. Biol. Chem.
BY
127
PYRIDOXAL-P
14. PENEFSKY, H. S. (1977) J. Biol. Chem. 252, 2891-2899. 15. OGAWA, H., AND FUJIOKA, M. (1980) J. Biol. Chem. 255, 7420-7425. 16. NEUJAHR, H., AND KJELLEN, K. (1980) Biochem-
istry 19,4967-4972. 17. MUHLRAD, A., AND TAKASHI, istry 20,6749-6754.
R. (1981)
Biochem-
6. NICHOLS,
18. GLAZER, A., DELANGE, R., AND SIGMAN, D. (1975) Chemical Modification of Proteins, pp. 131134, North-Holland/American Elsevier, New York.
7.
19. GOTO, Y., ZALKIN, H., KEIM, P., AND HEINRIKSON, R. (1976) J. Biol. Chem. 251, 941-949.
253,4659-4668.
8. 9. 10. 11.
B., MIOZZARI, G., VAN CLEEMPUT, M., BENNETT, G., AND YANOFSKY, C. (1980) J. Mol. Biol. 142, 503-518. Tso, J., HERMODSON, M., AND ZALKIN, H. (1980) J. Biol. Chem. 255, 1451-1457. Tso, J., AND ZALKIN, H. (1981) J. Biol. Chem. 256, 9901-9908. NAGANO, H., ZALKIN, H., AND HENDERSON, E. (1970) J. Biol. Chem. 245, 3810-3820. Tso, J., HERMODSON, M. A., AND ZALKIN, H. (1982) J. Biol. Chem. 257, 3532-3536. HARTMAN, S. C. (1963) J. Biol. Chem. 238,3036-
3047. 12. HENDERSON, E., NAGANO, H., ZALKIN, H., AND HWANG, L. (1970) J. Biol. Chem. 245, 14161423. 13. ZALKIN, H., AND KLING, D. (1968) Biochemistry
7, 3566-3573.
20. BLEILE, (1976)
D., JAMESON, J. Biol. C&m.
J.,
AND
HARRISON,
21. PAECH, C., AND TOLBERT, Chem. 253, 7864-7873.
N.
(1978)
22. BRAND, K., AND HORECKER, B. Biochem. Biophys. 123, 312-316. 23. AUTOR, A., AND FRIDOVICH, Chem. 245, 5214-5222.
241.4290-4292.
J.
(1968)
I. (1970)
24. HANSEN, B., LANE, R., AND DEKKER, J. Biol. Chem. 249, 4891-4896. 25. CASH, D., AND WILSON,
J.
251, 6304-6307.
I. (1966)
Biol. A&L
J. Biol. E. (1974)
J. Biol.
Chem.