Biochemistry 1989, 28, 43 1-437 Inoue, M., Kishimoto, A., Takai, Y., & Nishizuka, Y. (1977) J . Biol. Chem. 252, 7610-7616. Jaken, S., & Kiley, S . C. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 4418-4422. Kikkawa, U., Takai, Y., Minakuchi, R., Inohara, S . , & Nishizuka, Y. (1982) J . Biol. Chem. 257, 13341-13348. Kikkawa, U., Ono, Y., Ogita, K., Fujii, T., Asaoka, Y., Sekiguchi, K., Kosaka, Y., Igarashi, K., & Nishizuka, Y. (1987) FEBS Lett. 217, 227-231. Knopf, J. L., Lee, M.-H., Sultzman, L. A,, Kriz, R. W., Loomis, C. R., Hewick, R. M., & Bell, R. M. (1986) Cell (Cambridge, Mass.) 46, 491-502. Kubo, K., Ohno, S., & Suzuki, K. (1987) Nucleic Acids Res. 15, 7 179-7 180. Laemmli, U. K., & Favre, M. (1973) J . Mol. Biol. 80, 575-599. Le Peuch, C. J., Ballester, R., & Rosen, 0. M. (1 983) Proc. Natl. Acad. Sci. U.S.A. 80, 6858-6862. Ligeti, E., Doussicre, J., & Vignais, P. V. (1988) Biochemistry 27, 193-200. Lowry, 0. H., Rosebrough, N. J., Favor, A. L., & Randall, R.-J. (1951) J . Biol. Chem. 193, 265-275. McCaffrey, P. C., Rosner, M. R., Kikkawa, U., Sekiguchi, K., Ogita, K., Ase, K., & Nishizuka, Y. (1987) Biochem. Biophys. Res. Commun. 146, 140-146. McDonald, J. R., Groschel-Stewart, U., & Walsh, M. P. (1987) Biochem. J . 242, 695-705. Morel, F., Doussicre, J., Stasia, M.-J., & Vignais, P. V. (1985)
43 1
Eur. J . Biochem. 152, 669-679. Nishizuka, Y. (1984) Nature (London) 308, 693-698. Ohno, S., Kawasaki, H., Imajoh, S., Suzuki, K., Inagaki, M., Yokokura, H., Sakoh, T., & Hidaka, H. (1987) Nature (London) 325, 161-166. Ono, Y., & Kikkawa, U. (1987) Trends Biochem. Sci. (Pers. E d . ) 12, 421-423. Ono, Y., Kurokawa, T., Fujii, T., Kawahara, K., Igarashi, K., Kikkawa, U., Ogita, K., & Nishizuka, Y. (1986) FEBS Lett. 206, 347-352. Ono, Y., Fujii, T., Ogita, K., Kikkawa, U., Igarashi, K., & Nishizuka, Y. (1987) FEBS Lett. 226, 125-128. Parker, P. J., Coussens, L., Totty, N., Rhee, L., Young, S., Chen, E., Stabel, S., Waterfield, M. D., & Ullrich, A. (1986) Science (Washington, D.C.) 233, 853-859. Patterson, M. S . , & Greene, R. C. (1965) Anal. Chem. 37, 854-857. Pelosin, J.-M., Vilgrain, I., & Chambaz, E. M. (1987) Biochem. Biophys. Res. Commun. 147, 382-391. Rossi, F. (1986) Biochim. Biophys. Acta 853, 65-89. Stasia, M.-J., Dianoux, A.-C., & Vignais, P. V. (1987) Biochem. Biophys. Res. Commun. 147, 428-436. Tauber, A. I. (1987) Blood 69, 711-720. Walton, G. M., Bertics, P. J., Hudson, L. G., Vedvick, T. S., & Gill, G. N. (1987) Anal. Biochem. 161, 425-437. Yeng, A. Y., Sharkey, N. A,, & Blumberg, P. M. (1986) Cancer Res. 46. 1966-1971.
Mechanism of Inactivation of Alanine Racemase by P,P,P-Trifluoroalanine+ W. Stephen Faraci and Christopher T. Walsh* Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 021 15 Received June 8. 1988; Revised Manuscript Received August 24, 1988
The alanine racemases are a group of PLP-dependent bacterial enzymes that catalyze the racemization of alanine, providing D-alanine for cell wall synthesis. Inactivation of the alanine racemases from the Gram-negative organism Salmonella typhimurium and Gram-positive organism Bacillus stearothermophilus with P,P,P-trifluoroalanine has been studied. The inactivation occurs with the same rate constant as that for formation of a broad 460-490-nm chromophore. Loss of two fluoride ions per mole of inactivated enzyme and retention of [ 1-I4C]trifluoroalanine label accompany inhibition, suggesting a monofluoro enzyme adduct. Partial denaturation (1 M guanidine) leads to rapid return of the initial 420-nm chromophore, followed by a slower ( t l l Z-30 min-1 h) loss of the fluoride ion and I4CO2release. At this point, reduction by NaB3H4 and tryptic digestion yield a single radiolabeled peptide. Purification and sequencing of the peptide reveals that lysine-38 is covalently attached to the P L P cofactor. A mechanism for enzyme inactivation by trifluoroalanine is proposed and contrasted with earlier results on monohaloalanines, in which nucleophilic attack of released aminoacrylate on the P L P aldimine leads to enzyme inactivation. For trifluoroalanine inactivation, nucleophilic attack of lysine-38 on the electrophilic P-difluoro-a$-unsaturated imine provides an alternative mode of inhibition for these enzymes. ABSTRACT:
T e D-isomer of alanine is an important component of the peptidoglycan, which is the extensively cross-linked, rigid structure essential to the integrity of the bacterial cell wall. The alanine racemases, a group of pyridoxal 5’-phosphate (PLP)’ containing enzymes, provide bacteria with D-alanine ‘This work was supported in part by a grant from the National Science Foundation (PCM 8308969). W.S.F. was supported by a National Institutes of Health postdoctoral fellowship (GM 11471-02).
through the racemization of available L-alanine (Adams, 1976; Faraci & Walsh, 1988). Because of the importance of these enzymes for bacterial growth, they are attractive targets for antibacterial drug development. Abbreviations: PLP, pyridoxal 5’-phosphate; PMP, pyridoxamine 5’-phosphate; CHES, 2-(N-cyclohexylamino)ethanesulfonic acid; HEPES, N-(2-hydroxyethyl)piperazine-N’-2-ethanesulfonicacid; TPCK, L- 1-chloro-3-(4-tosylamino)-4-phenyl-2-butanone.
0006-2960/89/0428-043 1$01.50/0 0 1989 American Chemical Society
432
Biochemistry, Vol. 28, No. 2, 1989
Faraci and Walsh
Scheme I
H
H
*o H
Inactive Enzyme
Previous work has shown that inactivation of the alanine racemases from the Gram-negative bacterium Salmonella typhimurium (Badet et al., 1984; Esaki & Walsh, 1984), in which there are two distinct enzymes, dadB and a h (Wasserman et al., 1983), and the Gram-positive bacterium Streptococcus faecalis (Badet & Walsh, 1985) by the @-haloalanines (e.g., chloroalanine, fluoroalanine) proceeds through nucleophilic attack of nascent aminoacrylate on the PLP-lysine aldimine. This is shown in Scheme I. It appears that inactivation of a number of PLP-containing enzymes with @-substituted alanine analogues follows this mechanism (Likos et al., 1982; Ueno et al., 1982). The @-carbonof the enzymeinhibitor complex (1 in Scheme I) is not sufficiently electrophilic to be captured; hence, the active-site lysine undergoes transaldimination. The presence of a potent nucleophile (aminoacrylate) at the active site then leads to inactivation as the PLP cofactor is alkylated. The polyhalogenated alanine analogue @,@,@-trifluoroalanine is also known to be a potent inhibitor of PLP-containing enzymes (Silverman & Abeles, 1976, 1977). However, the mechanism of inhibition by trifluoroalanine is believed to be quite different from that observed for monohaloalanine. Although a great deal of work has been done investigating the inactivation mechanism of alanine racemase with the monohaloalanines (Badet et al., 1984; Roise et al., 1984; Esaki & Walsh, 1986), little has been done to determine the inactivation mechanism with @,@,@-trifluoroalanine (Wang & Walsh, 1981). In this paper, we report a detailed investigation of the mechanism of inactivation of the alanine racemase from the Gram-positive thermophile Bacillus stearothermophilus and Gram-negative S . typhimurium by trifluoroalanine. EXPERIMENTAL PROCEDURES
Materials. L- and D-alanine, NADH, CHES, HEPES, and trypsin (bovine pancreas; type I) were purchased from Sigma Chemical Co. Lactate dehydrogenase (10 mg/mL; 550 units/mg), D-amino-acid oxidase (5 mg/mL; 15 units/mg), and TPCK were from Boehringer-Mannheim Biochemicals. D,LTrifluoroalanine was purchased from Merck Schuchardt. TISAB (total ionic strength activity buffer) solution and fluoride standard were from Orion Research. Sodium [3H]borohydride (280 mCi/mmol) and potassium [*4C]cyanide(50 mCi/mmol) were purchased from Amersham. Centricon microconcentration filters ( M , cutoff 30 000) were purchased from Amicon. Synthesis of [ I 4 C ] Trifuoroalanine. This was synthesized by using a modified procedure of R. Silverman (personal
communication). Benzamide (3.0 g, 25 mmol) was dissolved in 30 mL of dimethoxyethane under argon. Boron trifluoride (60 pL of 40% in acetic acid) was added and stirred at 60 "C. Dry trifluoroacetaldehyde ethyl hemiacetal (20 mL) was added dropwise and the solution refluxed under argon for 1 h. Evaporation of solvent under reduced pressure led to 2,2,2trifluoro- 1-hydroxy-N-benzoylethylamineas product. Synthesis of the chloro derivative was achieved by dissolving 3 g (14 mmol) of the above product in 50 mL of dry dimethoxyethane upon which 3.6 g of phosphorus pentachloride was added. The solution was heated to 75 OC for 5 min at which time the solvent was removed by evaporation under reduced pressure. Purification by silica gel chromatography (methylene chloride as solvent) yielded 2,2,2-trifluoro- 1-chloro-Nbenzoylethylamine as product; 200 mg of the product (0.84 mmol) was dissolved in 5 mL of tetrahydrofuran to which triethylamine (1 mmol) was added. The mixture was filtered under argon to remove the precipitate. The flask containing the filtrate was connected to a flask containing a mixture of [14C]KCN and cold KCN. The filtrate was frozen, and both flasks were evacuated under vacuum. After thawing of the filtrate, 2.0 mL of 80% sulfuric acid was added to the KCN and the solid dissolved. Gentle heating led to generation of HCN, which can gain access to the filtrate flask that was in an ice bath. After 1 h, the ice bath was removed and the reaction mixture stirred for 14 h at room temperature. Evaporation of solvent under reduced pressure led to 2,2,2trifluoro-l-[14C]cyano-N-benzoylethylamine. Hydrolysis by refluxing in acid (6 N HC1) for 5 h led to product trifluoroalanine in 20% overall yield. Specific activity was calculated to be 0.35 mCi/mmol. Enzymes. The alanine racemases from the Gram-negative S . typhimurium (dadB and alr) and from B. stearothermophilus were purified as described previously (Faraci & Walsh, 1988). Analytical Methods. Enzyme activity was measured routinely in 0.1 M CHES buffer (pH 9.1) at 37 "C following the procedure of Badet et al. (1984) unless otherwise stated. Absorption spectra and activity readings were measured with a Hewlett-Packard diode array spectrophotometer. Radioactivity was measured by dissolving aliquots ( I 1 mL) into 5 mL of scintillation fluid and placing them in a Beckman LS1800 scintillation counter. Inactiuation Kinetics. The inactivation of alanine racemase by trifluoroalanine was measured at 25 OC in 0.1 M HEPES, pH 7.3, with trifluoroalanine (ranging in concentration from 5 to 40 mM) and racemase (0.5 pM for B. stearothermophilus, 0.35 pM for dadB, and 3 pM for a h ) . At various time intervals, aliquots were taken and activity was measured. Spectral Change and Release of Fluoride upon Inactivation of Racemase with Trifluoroalanine. The loss of fluoride ion was determined with a fluoride electrode (Model 96-09, Orion Research, Inc.), which was used in conjunction with an Orion Model 701A digital ionalyzer. A 1.0-mL solution containing alanine racemase from B . stearothermophilus (40-80 pM) and S . typhimurium (dadB, 6-30 pM; alr, 30-40 pM) was inactivated by addition of ca. 1 mg of trifluoroalanine, and the absorption spectra were recorded as a function of activity. Upon enzyme inactivation, a 300-pL aliquot was withdrawn and 300 pL of TISAB solution was added. Immersion of the fluoride electrode into the solution gave a reading that was stable in roughly 3 min. Calibration of the electrode with known standards allows correlation of the experimental reading with fluoride concentration. Measurement of fluoride concentration (at five
Biochemistry, Vol. 28, No. 2, 1989 433
Trifluoroalanine Inactivation of Alanine Racemase different enzyme concentrations) ensured accuracy of the result. Controls were performed in which fluoride concentration was measured in a similar incubation of enzyme with no trifluoroalanine and in a solution of trifluoroalanine with no enzyme added. Inactivation of Racemase by [l 4 C ] Trifluoroalanine. One milliliter of an 80 pM enzyme solution (B. stearothermophilus) was inactivated by the addition of 2 mg of [14C]triflu~r~lanine (0.35 mCi/mmol). The inactivated enzyme was separated from excess [ 14C]trifluoroalanine by Centricon filtration and washing with buffer until background level of radioactivity is reached. An absorption spectrum was taken to determine enzyme concentration, and an aliquot was assayed for radioactivity. Denaturation by addition of guanidine (to a final concentration of 4 M) or boiling (2 min) allowed determination of the fate of the radioactive label. Addition of guanidine (4 M) to an inactive enzyme solution led to a change in the absorption spectrum characteristic of denaturation as free PLP was released (A, = 390 nm). Centricon filtration allowed separation of enzyme from solute, and absorption spectra and radioactivity assays of both fractions were obtained. Acidification of the filtrate (i.e., flow-through fraction) with concentrated HC1 was followed by evaporation under reduced pressure. The residue was then dissolved in distilled water, and an absorption spectrum and radioactivity assay were taken. Heat denaturation led to an enzyme precipitate that was separated by centrifugation. An absorption spectrum and radioactivity assay of the supernatant solution and the precipitated enzyme, which was dissolved in 6 M guanidine, were taken. Acidification of the supernatant fraction, evaporation under reduced pressure, spectra, and radioactivity assays as described above were performed. Inactivation of Racemase by Trifluoroalanine and Reduction by Sodium [3H]Borohydride. One milliliter of a 60 pM enzyme solution (B. stearothermophilus) was inactivated by the addition of 2 mg of trifluoroalanine. At this time, 100 mg of guanidine (1 M final concentration) was added and the reaction incubated at 37 OC for 1 h. An absorption spectrum was taken of the partially denatured inactive enzyme species. This was followed by the addition of 2 mg of sodium [3H]borohydride (4 mCi/mmol) and 300 mg of guanidine (4 M final concentration), and incubation was continued for an additional 2 h. Excess radioactivity was separated from enzyme by Centricon filtration and washing with 4 M guanidine/0.3 M Tris/3 mM EDTA at pH 8. Reductive Alkylations, Trypsin Digest, and HPLC Peptide Purification. A total of 2 mg of inactive enzyme in guanidine/Tris/EDTA buffer as described above was alkylated with iodoacetamide and digested with TPCK-treated trypsin, and the radioactive peptide was purified by reverse-phase HPLC as described earlier (Badet et al., 1984). Peptide sequencing of the radioactive peptide was performed by W. Lane at the Harvard Microanalysis Facility, Cambridge, MA. Addition of Hydroxylamine to Inactive Enzyme. To 200 p L of the enzyme solution that had been inactivated with trifluoroalanine and reduced with sodium [3H]bor~hydrideas described above was added 200 pL of 4 M hydroxylamine (pH 8) and the solution incubated at 37 OC for 18 h. Enzyme was separated from excess small molecules by Centricon filtration and radioactivity measured in each fraction. RESULTS Inactivation of Alanine Racemase by Trifluoroalanine. Incubation of trifluoroalanine with the alanine racemases from S . typhimurium and B. stearothermophilus leads to timedependent inhibition; the kinetics of inactivation are shown
Table I: Kinetic Constants of S. typhimurium and B. stearothermophilus Alanine Racemase Inactivation by
Trifluoroalanine" ~
enzyme Ki(mM) kinan(min-I) B. stearothermophilus >lo0 >2.0 40f 10 0.15 f 0.04 dadB 65 f 10 0.08 f 0.02 a Ir Determined by double-reciprocal plots of inactivation rate against inhibitor concentraton.
3
0'4r 0.3
I " . ,/'
..,
&'-.. \
\i?. ,\\
%.-.-
0 360
I
I
I
I
400
440
480
520
Wavelength (nm)
1: Visible spectrum of alanine racemase after reaction with trifluoroalanine: Alanine racemase (28 pM) in buffer (0.1 M HEPES, pH 7.3); alanine racemase incubated with trifluoroalanineat 100% activity (-), 75% activity (- -), 57% activity (---), 27% activity (---), and 2% activity FIGURE
-
(.-.e).
in Table I. The values for Ki are very high, indicative of poor binding, due to either steric interference of the trifluoromethyl group at the active site or low concentration of zwitterionic trifluoroalanine,* which may be important in binding. Upon inactivation, a change in the absorption spectrum of the native enzyme is observed, concomitant with loss in enzyme activity, and is seen in Figure 1. A change in the A, from 41 8 nm (characteristic of PLP Schiff base formation) to a broad 460-490 nm suggests that the inactive enzyme species is different than the inactive enzyme complex formed upon inactivation of alanine racemase with the haloalanines (Badet et al., 1984; Roise et al., 1984), in which the 418-nm chromophore is replaced by a 325-nm chromophore, indicative of an unconjugated pyridoxyl group. The spectral change observed upon enzyme inactivation by trifluoroalanine is very similar to the absorption change of y-cystathionase, a PLPcontaining enzyme, upon inactivation by trifluoroalanine (Silverman & Abeles, 1977). Upon inactivation, 2 equiv of fluoride ion are released per mole of enzyme for the B. stearothermophilus racemase, and 5-10 equiv of fluoride ion are released per mole of enzyme for the S . typhimurium enzymes. These values are very similar to that obtained by Wang and Walsh (1981), using the Escherichia coli alanine racemase, suggesting that little (S. typhimurium) or no (B. stearothermophilus) turnover occurs prior to inhibition. The release of only two of the initial three fluorine atoms of trifluoroalanine in B. stearothermophilus racemase inactivation prompted us to probe this stoichiometry and its mechanistic constraints further. Inactivation with [I-l4C]Trifluoroalanine. Inactivation of the B. stearothermophilus alanine racemase with [ l-14C]trifluoroalanine showed that 1 mol of 14C-labelis associated per mole of enzyme. This information, along with that discussed above, allows one to construct a mechanism of inactivation The pK, of the amino group of trifluoroalanine is 5.8 (Wang & Walsh, 1981); thus, at pH 7.3, it is present (to 95%) as a carboxylate anion with an unprotonated amine.
434
Biochemistry, Vol. 28, No. 2, 1989
Faraci and Walsh
Scheme I1
H Enz-NH' II PLP
+
CF,&
CO;
-
"2
F H I
I
F- $- C - CO; AH+
-HF
-L
F,
2
c c - co; . .. .
II
c;H
CH
* O H
H
-1
2
x, c c - co; F'
I
NH t II
4 0 H H
as visualized in Scheme 11. Addition of trifluoroalanine to the resting enzyme leads to initial transaldimination, 1, followed by elimination of HF by a - H deprotonation and loss of fluoride ion from the resultant a-carbanion to give the P-difluoro-a,@-unsaturated imine complex, 2. Elimination of the second fluoride ion would occur concomitant with the enzyme inactivation process by nucleophilic attack of an enzyme group X on the olefinic terminus of the electrophilic P-difluoro-a,@-unsaturated imine. The resultant inactive complex, 3, would be covalently bound to the enzyme through the group X and would have extended conjugation, exemplified by the change in the absorption spectra upon inactivation to longer wavelength. Denaturation of Inactive Enzyme. Inactivation of the alanine racemase from B. stearothermophilus with [ 1-14C]trifluoroalanine leads to an enzyme species containing 1 equiv 14C-radiolabeland also still 1 equiv of fluoride. Denaturation by guanidine (4 M) or heat (boiling for 2 min) leads to a number of significant changes in the inactive enzyme complex. The first is the loss of the radioactive label from the enzyme. Upon denaturation, the label is absent from the enzyme fraction but is detected in the filtrate. Acidification of the supernatant and evaporation leads to complete loss of the label, suggesting that on denaturation the inactive enzyme complex (3 in Scheme 11) decarboxylates, releasing [ 14C]carbondioxide in the process. A second change that occurs is the elimination of 1 equiv of fluoride ion. Thus, a fluoride inventory shows that at this point all three fluoride atoms from the original trifluoroalanine have been released, the first two after formation of the inactive enzyme complex, while the third occurs only after protein denaturation. A third change is the release of free pyridoxal 5'-phosphate. This is detectable by the absorption at 390 nm, indicative of PLP, in which the absorption maximum is shifted upon addition of external amines (e.g., guanidine, alanine) to 41 5 nm, characteristic of Schiff base formation. Partial Denaturation and Reduction by Sodium 13H]Borohydride. Addition of sodium borohydride to the inactive enzyme complex (3, in Scheme 11) led to no change in the PLP
chromophore, suggesting that reduction of the Schiff base did not occur. The inactive enzyme complex is apparently resistant to Schiff base reduction, in contrast to the native enzyme. However, addition of 1 M guanidine to the inactive enzyme complex led to a rapid change (ca. 2-5 min) in the absorption maximum from 460-490 to 420 nm, suggestive of a partial denaturation, in which the active site is perhaps more accessible to solvent. Measurement of fluoride and 14C02upon this partial denaturation condition showed that 1 equiv each of fluoride and 14C02released. However, the rate of elimination for both fluoride and 14C02was much slower (ca. t I l 2 of 30 min-1 h) than the rate of PLP imine return, associated with partial denaturation, indicating that partial denaturation occurs first, followed by the release of fluoride and 14C02. A mechanism to explain the observed changes on denaturation is depicted in Scheme 111. Partial denaturation of the inactive enzyme complex may lead, by H 2 0 addition to the @-carbon of 3, to the hydrated species 4, characterized by a change in the absorption spectrum from the 460-490-nm maximum to the 420-nm maximum, identical with native enzyme. The rate for subsequent elimination of fluoride and 14C02(yielding species 5 and 6, respectively, in Scheme 111) were found to be similar ( t l l Z of 30 min-1 h). Addition of NaB3H4 to the partially denatured inactive enzyme complex, in contrast to the undenatured conditions, led to immediate reduction, characterized by a shift in the absorption maximum from 420 (PLP Schiff base) to 320 nm (PLP-reduced Schiff base). Isolation of the borohydride-reduced inactive enzyme complex showed 1 mol of 3H/mol of enzyme. Determination of the enzyme nucleophile X (Scheme 11) involved in the enzyme alkylative inactivation can be ascertained because NaB3H4reduction provides a label for the enzymic residue, even though the initial l-14C-label from trifluoroalanine has been lost (Scheme 111). Identification of Enzyme Nucleophile. Tryptic digest of the [3H]borohydride-reduced inactive enzyme complex and HPLC purification led to the isolation of a single radioactive peptide fragment. An absorption spectrum of the radioactive peptide showed a peak at 320 nm, characteristic of a PMP
Biochemistry, Vol. 28, No. 2, 1989 435
Trifluoroalanine Inactivation of Alanine Racemase Scheme I11
I H2 p;' 'co2 HO- F;- CH'
lYS38
+
H2
+
NH+
CH
CH
II
o@
+o H
a
4
I
NH + 11
II
H
'co,'
C - CH'
I
"+
yI
H
5 CHO
- 'co,
t
It
CH
"r" IYS38 " I
OsC-CH2
I
NH,+ I
+o H
one to propose an inactivation mechanism of the B. stearothermophilus alanine racemase with trifluoroalanine and to deduce the fate of the inactive enzyme species (3, in Scheme 11) upon denaturation, as seen in Schemes I1 and 111, where 3sLys-Ala-40Asn-Ala-TyrX (Scheme 11) is the active-site lysine residue. The inactivation sequence of labeled peptide" 3oAsp-Thr-His-Ile-Met-3sAla-Val-Val- mechanism proposed herein is similar to that proposed by XXXb-A1a-"Asn-Ala-TyrSilverman and Abeles (1977) in the inactivation of y-cysta" In the sequencing runs, the PMP-lysine adduct was not directly thionase by trifluoroalanine, where a lysine residue was simidentified. Residue containing the radioactivity. ilarly involved in presumed alkylative capture of a difluoro intermediate. derivative. An N-terminal sequence of the purified peptide Effect of Hydroxylamine on the Inactive Enzyme Species. fragment was obtained, and the sequencing data are sumIncubation of hydroxylamine with the inactive enzyme species marized in Table I1 and compared to the sequence predicted reduced with NaB3H4as described above led to less than 5% from the gene (Tanizawa et al., 1988). loss of radioactivity from the enzyme complex. As hydroxAnalysis of the data shows that the enzyme residue covaylamine can cleave thioestem (which would be present on lently bound to the inhibitor-PMP cofactor is lysine-38. This nucleophilic attack of cysteine), phenyl esters (on nucleophilic residue is the active-site lysine residue, which forms a Schiff attack of tyrosine), and imidazolides (on nucleophilic attack base with pyridoxal 5'-phosphate in the enzyme's resting state of histidine) readily, this result corroborates that an amide (Inagaki et al., 1986; Tanizawa et al., 1988). Although cycle (lysine) linkage is present in the reduced, inactive PMP enzyme 8 (lysine-38) in the sequencing reaction was the residue concomplex. taining all the radioactivity, this was only 10% of the total radioactivity of the applied sample. This is probably due to DISCUSSION the instability of the inactive enzyme species, as no radioactivity above background was detected in any other residue. As From the available information, we can arrive at an inacshown in Table 11, cycle 8 was a blank (shown as XXX), tivation mechanism of alanine racemase with trifluoroalanine, indicating that no amino acid was present. This is expected which is shown in Scheme 11. Incubation of the inhibitor with as the PMP-lysine adduct will give an abnormal HPLC trace. active enzyme leads to initial transaldimination from which Thus, identification of lysine-38 as the modified residue is by H F is subsequently eliminated to yield the @-difluoro-a,@comparison with the DNA sequence. The data presented allow unsaturated imine complex (2 in Scheme 11). This species is
Table 11: Amino Acid Sequence of Labeled Tryptic Peptides from NaB3H, Reduction of the B. stearothermophilus Alanine Racemase Inactivated by Trifluoroalanine from DNA sequence 3oAsp-Thr-His-Ile-Met-3SAla-Val-Val-
436
Biochemistry, Vol. 28, No. 2, 1989
Faraci and Walsh
Scheme IV
H
T +
HZN,
F/
H
c c - co; I
NH + II
I -
CH
* a potent electrophile, in which capture of an enzyme nucleophile occurs very rapidly. The enzymic nucleophile from the Gram-positive B. stearothermophilus alanine racemase is lysine-38, which is the active-site lysine. Although the enzyme nucleophile from the Gram-negative S. typhimurium alanine racemase has not been determined, similar absorption spectra (460-490-nm A,,) and fluoride loss upon inactivation are observed for all enzymes, suggestive of similar inactivation mechanisms. Thus, we predict that inactivation of the S. typhimurium alanine racemases by trifluoroalanine proceeds via the same mechanism as the B. steurothermophilus racemase. Work by Silverman and Abeles (1976,1977) has shown that inactivation of y-cystathionase by trifluoroalanine also proceeds via nucleophilic addition of (presumably) the active-site lysine on the @-difluoro-a,@-unsaturatedimine, formed on initial fluoride elimination. However, the structure of that inactive enzyme intermediate is different in that all three fluoride ions were released, giving a structure analogous to 5 in Scheme 111. The structure of the inactive alanine racemase species proposed herein (3 in Scheme 111) is unique, as @-monofluoro-a,@-unsaturatedimines are susceptible to facile hydration through Michael addition on the @-carbon. To explain the observations, we reason that on inactivation the active site becomes impermeable to solvent and small molecules (e.g., sodium borohydride), perhaps through a conformational change. Thus, a recurrent theme in racemase inactivation is seen; a change in the enzyme conformation is observed upon enzyme inactivation. Inactivation of the Gram-positive B. stearothermophilus alanine racemase by ( 1-aminoethyl)phosphonic acid (Ala-P) (Badet et al., 1986) leads to an Ala-P-PLP aldimine enzyme species that is resistant to borohydride reduction. It is believed that a conformational change occurs after initial Schiff base formation to give the stable, slowbinding inhibitor complex (Copie et al., 1988). With trifluoroalanine inactivation, a conformational change apparently accompanies enzyme inhibition to give a borohydride-resistant enzyme complex in which solvent accessibility also appears limited. The stability of proposed structure 4, to be formed by hydration of the inactive enzyme species on partial denaturation, is quite surprising. It is known that a-fluorohydrins undergo fluoride elimination spontaneously by collapse of the tetrahedral intermediate to give the ketone (Marletta et al., 1982; Walsh, 1983). One reason that may account for this unusual
o
H
behavior is that the enzyme interferes with the decomposition pathway by making the elimination less favorable. It is also possible, although unlikely, that fluoride ion elimination occurs readily but remains associated with the enzyme and dissociates only slowly. The inactivation of alanine racemase by the @-substituted monohaloalanine analogues (e.g., fluoroalanine, chloroalanine) and trifluoroalanine proceeds by two different mechanisms, visualized in Scheme IV. Analysis of the two mechanisms indicates that inactivation after formation of the cross-conjugated eneamino-PLP species (I in Scheme IV) can proceed by two routes, a and b. In the case where the @-carbonsubstituents are two hydrogens, the electrophilicity at the olefinic terminus is insufficient to trap the nearby nucleophile, the c-NH2 of the active-site lysine-38. Instead, it engages in normal nucleophilic attack on the aldimine carbon to initiate the normal transaldimination process involved in product release (route a). Inactivation is now an epiphenomenon that is a competition between eneamino acid release and its adventitious reaction as a nucleophile to capture the PLP-Lys,* resting aldimine (Scheme I). The keynote of this inactivation route is inactivator as nucleophile and no direct covalent linkage of enzyme to the three-carbon inactivator. In the case where the initial H F elimination leads to two fluorine substituents on the @-carbonof the eneamino-PLP (I) the partition is quite distinct. Now the lysine-38 amino group again acts as nucleophile, but now only by path b, where Michael-type attack on the highly electron deficient difluoroeneamino terminus is apparently exclusively favored over path a. Path b then leads to direct covalent attachment of the enzyme-Lys38-NH2 group to the three-carbon inactivator by its capture as an electrophilic fragment rather than a nucleophilic fragment (path a). The differential electrophilicity of two F vs two H substituents at the @-carbonof the eneamino-PLP complex completely reroutes the a / b partition. These two very diffeent modes of inactivation are indicative of the finely balanced chemistry accessible in PLP-containing enzymes. The number of turnovers per inactivation event is also dependent on which inactivation mechanism is followed. For example, in path a, the alanine racemase from S. typhimurium (dudB) will turn over fluoroalanine 800 times (to give fluoropyruvate) prior to inactivation (Badet et al., 1984). On trifluoroalanine inhibition, roughly three turnovers (to give difluoropyruvate) occur per inactivation. The difference in the partition ratio (800/3 -260/1) most likely may represent a difference in the reactivity of the two killing species at the
Trifluoroalanine Inactivation of Alanine Racemase
Biochemistry, Vol. 28, No. 2, 1989 437 Registry No. PLP, 54-47-7; D,L-trifluoroalanine, 17463-43-3; [ 14C]-j3,j3,j3-trifluoro-~~-alanine, 117624-87-0;benzamide, 55-21-0; trifluoroacetaldehyde ethyl hemiacetal, 433-27-2; 2,2,2-trifluoro-1hydroxy-N-benzoylethylamine, 1 17606-59-4; 2,2,2-trifluoro-1chloro-N-benzoylethylamine,117606-60-7; 2,2,2-trifluoro-l-[14C]cyano-N-benzoylethylamine, 1 17606-61-8; lysine, 56-87- 1; alanine racemase, 9024-06-0.
FIGURE 2: Proposed orientation of the active-site lysine-38 r-amino group for a-H abstraction, PLP-aldimine attack, and &carbon attack.
active site, the P-difluoro-a,@unsaturated imine being a more potent inactivator (as an electrophile) than the nascent paminoacrylate reacting with opposite polarity (as a nucleophile). The ability of the active-site lysine to add covalently to the 8-carbon of the &difluoro-a,P-unsaturated imine species suggests a substantial amount of mobility of this residue. Although there is no direct evidence to favor a one- or two-base mechanism in racemase catalysis (Faraci & Walsh, 1988; Soda et al., 1986), total incorporation of label only at lysine-38 and lack of ability of external nucleophiles to release 3H-label is supportive of capture of only one nucleophile and, by extension, of a one-base mechanism in which the active-site lysine is the base. A schematic representation of the racemase active site showing the proposed versatility of lysine-38 is seen in Figure 2. The ability of the lysyl group to attack the P-carbon and 4'-carbon on PLP (Scheme IV) as well as to act as proposed catalytic base to abstract the a-hydrogen suggests that catalysis is a dynamic process in which the enzyme base and PLP cofactor are flexible. Thus, a mechanism similar to that proposed by Henderson and Johnston (1976) for racemase catalysis may be operative. The ability of a PLP-containing enzyme to use the active-site lysine as a single base to conduct proton transfer is not unprecedented; asparate aminotransferase (Torchinsky, 1986) uses the active-site lysine as a single base in catalysis. ACKNOWLEDGMENTS We are grateful to M. Distefano, K. Duncan, and L. Zawadzke for helpful discussions. We thank Dr. W. Lane at the Harvard microanalysis facility for peptide sequencing and Dr. R. Silverman for help in the synthesis of [ 14C]trifluoroalanine.
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