mechanism of the irreversible inactivation of mouse ornithine

THE ~OLIRNAI. OF BIOLOCICAI. CHEMISTRY ‘ ( 1 1992 hy The American Society for Biochemistry and Molerular B101oc.y. Inc.

Vol. 267, No. I , Issue of January 5, pp. 150-158, 1999 Printed ~n U S A .

Mechanism of the Irreversible Inactivation of Mouse Ornithine Decarboxylase by a-Difluoromethylornithine CHARACTERIZATION OF SEQUENCES AT THE INHIBITOR AND COENZYME BINDING SITES*

(Received for publication, August 12,1991)

Richard PoulinSg, Li LuS, Bradley Ackermannll, Philippe Beyll, and Anthony E. Peggfll From the $Departments of Cellular and Molecular Physiology and Pharmacology, The Milton S. Hershey Medical Center, Pennsylvania State University College of Medicine, Hershey, Pennsylvania I7033 and the TMarion Merrell Dow Reseaich Institute, Cincinnati, Ohiob52I5

Mouse ornithine decarboxylase (ODC) was expressed in Escherichia coli and the purified recombinant enzyme used for determination of the binding site for pyridoxal 5”phosphate and of the residues modified in the inactivation of the enzyme by the enzyme-activated irreversible inhibitor, a-difluoromethylornithine (DFMO). The pyridoxal 5”phosphate binding lysine in mouse ODC was identified as lysine 69 of the mouse sequence by reduction of the purified holoenzyme form with NaB[“HI4 followed by digestion of the carboxymethylated protein with endoproteinase Lys- C, radioactive peptide mapping using reversed-phase high pressure liquid chromatography and gas-phase peptide sequencing. This lysine is contained in the sequence PFYAVKC, which is found in all known ODCs from eukaryotes. The preceding amino acids do not conform to the consensus sequence of SXHK, which contains the pyridoxal 5”phosphate binding lysine in a number of other decarboxylases including ODCs from E. coli. Using a similar procedure to analyze ODC labeled by reaction with [5-’*C]DFMO, it was found that lysine 69 and cysteine 360 formed covalent adducts with the inhibitor. Cysteine 360, which was the major adduct accounting for about 90% of the total labeling, is contained within the sequence -WGPTCDGL(I)D-, which is present in all known eukaryote ODCs. These results provide strong evidence that these two peptides form essential parts of the catalytic site of ODC. Analysis by fast atom bombard- ment-mass spectrometry of tryptic peptides containing the DFMO-cysteine adduct indicated that the adduct formed in the enzyme was probably the cyclic imine S- ((2-(l-pyrroline))methyl)cysteine. This is readily oxidized to S-((2-pyrrole)methyl)cysteine or converted to S-((2-pyrrolidine)methyl)cysteine by NaBH4 reduction. This adduct is consistent with spectral evidence showing that inactivation of the enzyme with DFMO does not entail the formation of a stable adduct between

* This research was supported in part by Grants CA-18138 and CA-37606 from the National Institutes of Health. Protein sequencing was made possible by National Science Foundation Biological Facility Center Grant DIR 8804758. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Supported by a postdoctoral fellowship from the Medical Re- search Council of Canada. Present address: MRC Group in Molecular Endocrinology, Lava1 University Medical Center, Quebec, Canada GIV 4G2.

11 To whom correspondence should be addressed Dept. of Cellular and Molecular Physiology and Pharmacology, P. 0. Box 850, Hershey Medical Center, Pennsylvania State University College of Medicine, Hershey, PA 17033.

the pyridoxal 5’-phosphate, the enzyme, and the inhibitor.

L-Ornithine decarboxylase (ODC)’ is an important enzyme which, in mammalian cells and many other eukaryotes, is essential for the production of putrescine, the diamine pre- cursor of the polyamines. The activity of ODC is very rapidly and greatly changed in response to stimuli affecting cell growth and polyamine content, and the underlying biochemical mechanisms responsible for these changes have been the subject of extensive investigation (1-3). The structure and enzymatic properties of ODC have received much less atten- tion although recent studies in which the cDNA has been cloned have allowed the derivation of the amino acid sequences for ODCs from a number of sources including mouse (4-6), human (7), rat (8,9), hamster (lo), Xenopus laeuis (ll), Trypanosoma brucei (12), Saccharomyces cerevisiae (13), and Neurospora crassa.?

The eukaryotic ODC sequences show very little, if any, similarity to those of the Escherichia coli biosynthetic and biodegradative ODCs (14-16) and the Lactobacillus sp.30a ODC (17). However, comparisons of the eukaryotic ODC sequences show a remarkable similarity between ODCs from these sources with more than 90% identity between the mammalian proteins and an 81% (Xenopus), 69% (Trypanosoma), 42% (Neurospora), and 40% (yeast) identity between these proteins and the murine ODC over the common core region of the enzyme. Little information is available concerning the amino acids forming the active site of ODC. Complete loss of enzymatic activity of the mouse ODC occurs with the mutation to alanine of either His-197 or Lys-169 (18), and an inactive hamster ODC mutant was found to be caused by a change of glycine to aspartic acid at position 381 (19). Trun- cation of the mouse ODC at the carboxyl end to remove the terminal 37 amino acids did not affect the activity (18, 20). Even the site of binding of the pyridoxal 5”phosphate (PLP) cofactor was not known, and eukaryotic ODC does not contain the consensus sequence of -SXHK- which includes the PLP- binding lysine in many PLP-dependent enzymes including the E. coli ODCs (16, 21).

The abbreviations used are: ODC, ornithine decarboxylase (EC 4.1.1.17); DFMO, D,L-a-difluoromethylornithine; PLP, pyridoxal 5’- phosphate; IPTG, isopropyl 0-D-thiogalactopyranoside; P-pyridoxyl, phosphopyridoxyl; PTH, phenylthiohydantoin; RP-HPLC, reversed- phase high pressure liquid chromatography; FAB, fast atom bom- bardment; MS, mass spectrometry.

L. J. Williams, G. R. Barnett, J. L. Ristow, J. Pitkin, M. Prerriere, and R. H. Davis, unpublished sequence of N. crassa ODC (personal communication).

150

Structure of Mouse Ornithine Decarboxylase 151

DFMO was designed as an enzyme-activated irreversible inhibitor of ODC (22-24). As predicted, incubation of the eukaryote enzyme with DFMO leads to an irreversible loss of enzyme activity. The inhibition of T. brucei ODC is of major pharmacological importance since African sleeping sickness caused by this organism is very effectively treated by this drug, which is now in clinical usage for this purpose. DFMO may also have therapeutic potential for a number of other illnesses caused by protozoans and for diseases involving abnormal cellular proliferation, including cancer (25-27). As predicted from the proposed mechanism (22-24), the inactivation of mouse ODC by DFMO involves the decarboxylation of DFMO by the enzyme and the stoichiometric binding of a metabolite to the protein (28, 29). In the present paper we have determined the amino acid residues in mouse ODC which are involved in the covalent binding of DFMO, determined the structure of the major adduct, and located the lysyl residue forming a Schiff base with PLP. These results provide addi- tional information on the mechanism of action of DFMO and on the active site of ODC.

EXPERIMENTAL PROCEDURES

Materiak-~-[l-’~C]Ornithine (52 Ci/mol) and NaB[’’H], (490 Ci/ mol) were obtained from Du Pont-New England Nuclear. [5-I4C] DFMO (60 Ci/mol) was obtained from the Amersham Corp. Unla- beled DFMO was produced by the Marion-Merrell Dow Research Institute (Cincinnati, OH). N‘-phosphopyridoxyl-N“-tert-butyloxy- carbonyllysine, synthesized as described (30), was generously pro- vided by Dr. s. F. Yang (University of California, Davis). RP-HPLC- purified trypsin from bovine pancreas (sequencing grade, essen- tially (<0.01%) chymotrypsin free) and endoproteinases Lys-C (EC 3.4.21.50) and Glu-C (protease V8, EC 3.4.21.19) were obtained from Boehringer Mannheim. Isopropyl P-D-thiogalactopyranoside (IPTG), ampicillin (sodium salt), pyridoxamine 5’-phosphate, PLP, NaCNBH.,, CF:,COOH, and iodoacetic acid were purchased from Sigma. Sequenase 2.0 and guanidine hydrochloride (Ultrapure) were purchased from the U. S. Biochemical Corp. T4 DNA ligase was purchased from New England Biolabs (Beverly, MA) and dithiothreitol from Calbiochem. Brij 35 (30% (w/v) solution) was obtained from Technicon Instruments Corporation (Tarrytown, NY). NaBH, was supplied by Aldrich. E. coli strain EWH331 (31) was a generous gift from Drs. H. and C. W. Tabor, NIDDK, Bethesda, MD.

Construction of the PIN-ODC-5 Expression Vector-A plasmid expressing the mouse ODC in E. coli was constructed by introducing an EcoRI site close to the initiation codon of the mouse cDNA and using this site to insert the cDNA into the PIN-111 expression vector. This vector uses the lpp gene promoter in a plasmid which also contains the lac promoter operator fragment so that expression re- quires the addition of a lac inducer such as IPTG (32). The construction was carried out as follows. The plasmid pGEM-ODC, which contains the mouse cDNA (18), was used for site-directed mutagenesis to introduce an EcoRI site at positions 22-26 (relative to the A of the initiation codon). Oligonucleotide-directed mutagenesis was carried out as described (18) using a modification of the method described by Kunkel(33) and the mutagenic primer 5”GTGGCAGT- CGAATTCGTCCTTAGTAAAGCTGC-3’ (mismatches underlined). A plasmid containing the desired mutation was identified by cutting with EcoRI and then sequenced to confirm that the correct change was present. This plasmid, pGEM-ODC-M6 was then digested with EcoRI and BamHI, and the 1.6-kilobase fragment, which contains the ODC cDNA, was isolated and ligated into plasmid pIN-III-A2 which had been cleaved with EcoRI and BamHI at the multiple cloning site. The resulting plasmid PIN-111-ODC did lead to expression of mouse ODC when grown in E. coli, but to improve the level of expression, the lpp promoter in this vector was replaced with the stronger mutated lpp” ’ promoter (32). This was accomplished by cleavage of PIN-111-ODC with XbaI and BamHI to generate a fragment that contains the ODC insert and the 5’ region between the XbaI site and the initiation codon. This fragment was then ligated into pIN-III-lpp”~5”A3 (32) which had been cut with XbaI and BamHI. The final construct termed PIN-ODC-5 was used for expression of mouse ODC in E. coli EWH331, a strain that does not contain any endogenous ODC activity because of mutation in the spec gene (31).

Expression of Recombinant Mouse ODC-E. coli EWH331 trans-

formed with the PIN-ODC-5 vector was inoculated into 10 ml of a modified M9 medium containing 50 pg/ml ampicillin, 1.5% (w/v) casamino acids (Difco), 1.1 mM D-ghCOSe, 0.1 mM CaCh 1 mM MgS04, 1 mM thiamine HCI, and M9 salts (42 mM Na2HP04, 22 mM KH,PO,, 19 mM NH4CI, 8.5 mM NaCI) and grown a t 37 “c for 12-18 h in a 50-ml polypropylene conical centrifuge tube. Aliquots of the bacterial suspension (2.5 ml) were then added to 250 ml of modified M9 medium in 1-liter conical flasks and grown a t 37 “C with vigorous agitation (225-250 rpm) until the AsuS was about 0.4-0.5. IPTG (0.5 mM) was then added and the culture allowed to grow to an ASS of 1.5-2.0. Cells were harvested in round-bottom centrifuge bottles (4,400 X g for 10 min at 4 “C), resuspended with 36 ml of buffer A (25 mM Tris-HCI, pH 7.5, 0.1 mM EDTA, 2.5 mM dithiothreitol, 0.02% (w/v) Brij 35)/liter of culture, frozen in liquid NS, and stored a t -70 “C until use for enzyme purification.

Purification of Recombinant Mouse ODC-Unless otherwise indicated, all steps were performed a t 0-4 “C. The frozen cell suspension was thawed at room temperature and sonicated for 5 min in an ice- water bath using a Heat Systems-Ultrasonics Sonicator W 225R (0.5- s pulses at the maximum energy for a microtip). Solid streptomycin sulfate ( I%, w/v) was added to the cell homogenate, and after gentle magnetic stirring for 15 min, a crude enzyme extract was obtained by centrifugation (15,000 X g for 30 min). The pellet was discarded, and the supernatant was subjected to (NH4)sS0, fractionation (34). The protein fraction precipitating between 30 and 50% saturation with (NH&SO, was collected by centrifugation a t 10,000 X g for 10 min, dissolved in buffer A to a final volume of 2.5 ml, and desalted using a PD-10 gel filtration column (Pharmacia LKB Biotechnology Inc.) equilibrated with buffer A. The column eluate containing the protein fraction (3.5 ml) was then directly applied to a pyridoxamine 5’- phosphate-agarose affinity column (2.5 X 10 cm), prepared from Affi- Gel 10 resin (34), and preequilibrated with buffer A a t a flow rate of 4 ml/h. The column was washed with at least 6 column volumes of buffer A a t 16 ml/h, and recombinant mouse ODC was then eluted with buffer A containing 50 FM PLP at 16 ml/h, collecting 8-ml fractions. The fractions containing most (295%) of the ODC activity were pooled and concentrated by ultrafiltration using a Diaflo YM- 10 membrane to a 3-5-ml final volume.

Identification of the P-pyridoxyl-lysine Residue in Recombinant Mouse ODC-During all the following operations, the purified enzyme solution was protected from light to reduce photodecomposition of the cofactor and photooxidation of tryptophan residues. Immediately after purification by affinity chromatography, the concentration of PLP in the ODC preparation (3.1 mg) was lowered to 10 PM (determined using an extinction coefficient of 5.4 X 10:’ at 412 nm) by dilution with buffer A and the volume brought down to approximately 1 ml by ultrafiltration using a Centricon-10 microconcentrator (Ami- con, Beverley, MA). Solid NaBH, (0.4 mg) was mixed with an equal mass of NaB[’H], (5 mCi) in a glass vial to which the enzyme solution was then added, and the reaction was allowed to proceed for 45 min a t room temperature. The reaction mixture was then dialyzed against 1 liter of buffer A followed twice by 1 liter of buffer A in which the EDTA was increased to 2 mM. Guanidine hydrochloride was added to 6 M, and the buffer concentration was adjusted to 250 mM Tris- HCI, pH 8.5, 2 mM EDTA, 10 mM dithiothreitol. The enzyme was then incubated for 60 min a t 37 “C under an Nr atmosphere. Carbox- ymethylation of cysteine residues was then performed (35) with 20 mM sodium iodoacetate for 15 min at room temperature under an N, barrier, and the reaction was stopped by adding 35 mM 2-mercapto- ethanol. The carboxymethylated protein was dialyzed against 3 X 2 liters of Lys-C digestion buffer (Tris-HCI, pH 8.5, 1 mM EDTA, 5% (v/v) acetonitrile). After dialysis, the reduced, carboxymethylated ODC (2.2 mg), which had a total radioactivity of 1.6 X 10’ dpm, was digested with endoproteinase Lys-C for 18 h a t 37 “C, using a 1:25 (protease/substrate) mass ratio. The Lys-C-generated peptides were analyzed by RP-HPLC with a Beckman HPLC system model llOA and a Bio-Rad Hi-Pore RC-304 column (250 X 4.6 mm), using a linear gradient of 0-80% acetonitrile in 60 min, in the presence of 0.1% (v/v) CF:COOH. The flow rate was 1.5 ml/min. Peptides were detected by monitoring absorbance of the column eluate a t 215 nm, and the UV-absorbing fractions were collected manually and analyzed for radioactivity. The single “H-containing peptide fraction was concentrated by centrifugation under vacuum using a Speed-Vac concentrator (Savant Instruments, Farmingdale, NY). The radioactive peptide was then sequenced using an Applied Biosystems 477A Protein Sequencer. Fractions corresponding to 30% of the volume injected at each degradation cycle were collected and analyzed for radioactivity.

The identity of the labeled residue was determined by comparison

152 Structure of Mouse Ornithine Decarboxylase with authentic N"pyridoxy1-lysine in a paper chromatography system. N"phosphopyridoxyl-N"-tert-butyloxycarbonyIlysine, as well as 230 pg of the carboxymethylated NaB['HH],-reduced ODC holoenzyme were hydrolyzed in 6 M HCI for 24 h a t 105 "C, and the hydrolysis products were dissolved in 30 p1 of water and analyzed by paper chromatography using 1-butanol/pyridine/acetic acid/water (30:20:6:24, v/v) for development (30). The position of M-pyridoxyl- lysine (R, = 0.12) was identified by its UV fluorescence, and the radioactivity of the hydrolysis products was measured by liquid scin- tillation spectrometry of segments cut from the paper strip.

Identification of the DFMO Binding Sites in Recombinant Mouse ODC-The concentrated purified enzyme (7-8 p ~ , calculated using a subunit M, of 50,730) obtained after affinity chromatography was supplemented with PLP to obtain a final concentration of 40 p~ of the coenzyme (as measured by its A,J. ODC was then allowed to react with a 20-fold molar excess of [5-"C]DFMO for 1 h at 37 "C, resulting in 298% inactivation of the enzyme. The enzyme solution was concentrated to 0.8-1.2 ml using a Centricon-10 microconcentrator and dialyzed for 24 h against 3 X 500-ml portions of buffer A.

The DFMO-enzyme adduct was then allowed to react for 2 h a t room temperature with NaBH, (I mg/ml) as described by Hayashi et al. (36). After dialysis against 1 liter of buffer A, and then 2 X 1-liter changes of 25 mM Tris-HC1, pH 8.5, 2 mM EDTA, 10 mM dithiothreitol, the enzyme was reduced and carboxymethylated, digested with endoproteinase Lys-C, the peptides analyzed by RP-HPLC, and peptide sequencing performed on the labeled peptides as described above. The minor labeled fragment found in the Lys-C digest (peptide 24), which was eluting a t 38.4% (v/v) acetonitrile, was purified further prior to sequencing by separation on the Bio-Rad RC-304 column using a two-step gradient (from 0 to 33.6% (v/v) acetonitrile in 25 min, followed by 33.6 to 40% (v/v) acetonitrile in 35 min, in the presence of 0.1% (v/v) CF,,COOH) and a flow rate of 1.5 ml/min.

A portion of the major labeled peptide (peptide 29) obtained from the endoproteinase Lys-C digest was evaporated to dryness in a Speed-Vac, and digested further with endoproteinase Glu-C. A ratio of approximately 1:20 (w/w, enzyme/substrate) was used for digestion, which was allowed to proceed for 14 h a t 37 "C in 50 mM sodium phosphate, pH 7.8 (37). The resulting labeled peptide was then sequenced and the radioactivity released a t each degradation cycle determined.

Characterization of the Adduct Formed upon Reaction of DFMO with the Cys-360 Residue by FAB-MS-Recombinant mouse ODC (2.2 mg) was purified, reacted with unlabeled DFMO using a 1:20 ratio of enzyme to inhibitor, and the adduct was reduced with NaBH, as described above. After carboxymethylation of the reduced protein, the enzyme solution was dialyzed against 2 X 2-liter changes of trypsin digestion buffer (100 mM Tris-HCI, pH 8.5, 10% (v/v) acetonitrile) and subjected to proteolytic digestion with trypsin for 16 h a t 37 "C a t a 1:40 mass ratio of trypsin to ODC protein, in the presence of 1 mM CaCL The tryptic peptides were separated by RP-HPLC on a CIx reversed-phase column (Bio-Rad Hi-Pore RC-318,250 X 4.6 mm) using a linear gradient of 0-50% (v/v) acetonitrile over 60 min, followed by a linear gradient of 5040% (v/v) acetonitrile over 10 min, in the presence of 0.1% (v/v) CF:COOH. The peptides containing the DFMO-derived adduct were identified from the retention times observed for the labeled peptides generated by the tryptic digestion of ["CIDFMO-labeled ODC processed under identical conditions. The HPLC fractions containing these peptides were collected manually into glass test tubes, the sample was neutralized by the addition of 25 mM NH,HCO:, from a 1 M stock solution, pH 8.0, and the fractions were dried down with a Speed-Vac concentrator. Sam- ples were redissolved in 30 p1 of acetonitrile/water (30:70, v/v) prior to analysis by FAB-mass spectrometry and amino acid analysis.

FAB mass spect,ra were acquired using a ZAB2-SE double-focusing mass spectrometer (VG Analytical) at a mass resolution of 2,200 (10% valley). Ionization was accomplished by a cesium ion source operating at an anode potential of +30 kV. The ion source acceleration potential was +8 kV. The magnet was scanned from m/z 2,350 to 1,400 using an exponential profile at 100 s/decade to give a total scan time of about 20 s. Sample preparation for FAB consisted of applying a few p1 of the peptide solution to the FAB probe tip followed by the addition of 1-2 pl of a freshly prepared FAB matrix composed of thioglycerol, dithiothreitol, dithioerythritol (6:5:1, w/w/w).

Five-pl aliquots of the peptide solutions used for FAB-MS were taken for amino acid analysis using an Aminoquant AA Analyzer (Hewlett-Packard). This system combines precolumn derivatization with o-phthalaldehyde and 9-fluorenylmethyl chloroformate with gra- dientRP-HPLC and fluorescence detection (38). One hundred p1 of

0.1 N HC1 was added to each sample prior to hydrolysis which occurred in uacuu for 24 h a t 105 "C using 6 M HCI.

Spectrophotometric Analysis of ODC Inactivation by DFMO-Pu- rified recombinant mouse ODC (300 pg) in buffer A containing 50 p M PLP was loaded on a PD-10 gel filtration column and eluted with PLP-free buffer A. The volume was then reduced to 1.3 ml using a Centricon-10 microconcentrator, and 100 p l of this solution was transferred to a quartz microcuvette for spectrophotometry. Ultravi- olet and visible spectra of this ODC solution (5 p ~ ) were then obtained using a Beckman DU-65 spectrophotometer. The spectral changes resulting from the reaction of DFMO with ODC were studied by obtaining spectra (280-540 nm) of the enzyme solution a t various intervals after the addition of 100 p~ DFMO directly to the cuvette. The filtrate from the ultrafiltration step used to concentrate the enzyme was used as a blank for the spectrophotometric measure- ments.

The stability of PLP binding to the DFMO-inactivated ODC was studied by comparing its absorption spectrum with that of the apoenzyme by a modification of a published method (36,39). All operations were performed in the dark to minimize photodecomposition of PLP. First, 140-pg portions of a 5 p~ solution of recombinant mouse ODC in buffer A containing 50 p~ PLP were incubated for 60 min at 37 "C in the presence or absence of 100 p~ DFMO and then dialyzed for 24 h against 3 X 500-ml portions of PLP-free buffer A. After obtaining absorption spectra of the dialyzed enzyme, L-cysteine HC1 in sodium phosphate buffer, pH 8.0, was added to a final concentration of 50 mM L-cysteine and inorganic phosphate. The mixture was incubated for 60 min at 25 "C under an N, atmosphere and then dialyzed overnight at 4 "c against 500 ml of 100 mM L-cysteine HCI, 100 mM sodium phosphate, pH 8.0, and then 2 X 500 ml of buffer A. The absorption spectra were taken after concentration to 750 pl with a Centricon-10 microconcentrator.

Miscellaneous Procedures-Protein hydrolysis was performed for 24 h in an evacuated flask with phenolic 6 N hydrochloric acid. Free amino acids were then derivatized with phenyl isothiocyanate and analyzed with a PicoTag (Waters) HPLC system (40). ODC activity was determined by measuring the release of "CO, from L-[~-"C] ornithine during a 30-min assay a t 37 "C (28). One unit of ODC activity is defined as the amount of 14C02 produced in pmol/min. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis using 10% (w/v) acrylamide was performed according to the method of Laemmli (41). Protein was determined by the method of Bradford (42) using bovine serum albumin (fraction V; Miles Laboratories, Elkhart, IN) as standard.

RESULTS

Expression and Purification of Recombinant Mouse ODC- The level of expression of recombinant mouse ODC in E. coli EWH331-PIN-ODC-5 transformants after stimulation with IPTG was sufficiently high (about 3-6% of the total soluble protein) that ODC of sufficient purity for sequencing was obtained after affinity chromatography of the fraction of the crude soluble proteins precipitating between 30 and 50% (NH,),SO, on a pyridoxamine 5'-phosphate-agarose column. Large scale preparations typically showed a recovery of the order of 80-90% at the affinity chromatography step. The specific activity of the purified recombinant mouse ODC was 48 & 7 units/mg of protein, very similar to that reported for the homogeneous enzyme purified from mouse kidney (34). Thus, although the expression plasmid construction results in an alteration in the amino terminus of the enzyme (MSSFTKD- being changed to MKGK- in the recombinant enzyme), the catalytic activity of the recombinant enzyme is not significantly affected by the modification. This is consistent with the lack of conservation of this amino-terminal region between known ODC sequences.

Identification of the PLP-binding Residue in Mouse ODC- Recombinant mouse ODC holoenzyme reduced with NaB- [:'HI4 contained approximately 0.7 mol of PLP/mol of enzyme subunit, assuming the incorporation of one hydrogen atom/ reduced Schiff base. This is in reasonable agreement with the expected value of 1.0 since the possible isotope effect in the reduction step is unknown. Upon acid hydrolysis of the


NaB[3H],-reduced holoenzyme, a discrete fluorescent product (excitation h = 300 nm) was found by paper chromatography, which had the same RF as authentic M-pyridoxyl-lysine in paper chromatography and which contained most of the radioactivity recovered on the chromatogram (results not shown).

A single radioactive peptide was detected by RP-HPLC mapping of the Lys-C digest of the NaB[3H]q-red~ced mouse ODC (Fig. 1). Automated sequencing of the first 20 residues of this peptide indicated the presence of a single amino acid at each cycle and matched the predicted amino acid sequence of mouse ODC extending from Ala-58 to Val-77 (Fig. 2) (4- 6), except for Lys-69, which was not detected as a PTH- derivative at the corresponding position of the 3H-labeled peptide. Radioactivity was released at this position starting from the 12th cycle of degradation of this peptide (Fig. 2), clearly indicating that Lys-69 is indeed the PLP-binding residue in mouse ODC and that in this digest it is resistant

0 10 20 30 40 50 % Acetonitrile

FIG. 1. RP-HPLC analysis of NaB[3H]4-reduced ODC holoenzyme after digestion by endoproteinase Lys-C. Purified recombinant mouse ODC (3.1 mg) was reduced with NaB[3H], and carboxymethylated as described under "Experimental Procedures." An aliquot of this preparation (375 pg) was digested with 15 pg of endoproteinase Lys-C and then analyzed by RP-HPLC using a 0- 80% (v/v) acetonitrile gradient in 0.1% (v/v) CF3COOH on a C4 reversed-phase column. The and radioactivity profiles of the resolved peptides, which were collected manually, are shown on the right and left ordinates, respectively. The absorbance range was increased from 0-1.0 to 0-2.0 where indicated by the arrow. Symbols (0) correspond to the total radioactivity present in the corresponding peptide fraction.

1 0 A L P R V T P F Y A Y X C N D S R A I V

I ' # ' I ' / ' t ' I ' I ' I ' I ' I

2 9 -

0 8 - 0 >

5 7 -

._ 5 6 - > ._ z 5 -

2 - s 4 -

"x 2 - r

1 z l -

o " ' 1 ' " ' "

0 2 4 6 8 1 0 1 2 1 4 16 1 8 20

Cycle number

1 0 A L P R V T P F Y A Y X C N D S R A I V

I ' # ' I ' / ' t ' I ' I ' I ' I ' I

2 9 -

0 8 - 0 >

5 7 -

._ 5 6 - > ._ z 5 -

2 - s 4 -

"x 2 - r

1 z l -

o " ' 1 ' " ' "

0 2 4 6 8 1 0 1 2 1 4 16 1 8 20

Cycle number

FIG. 2. Partial amino acid sequence and site of labeling of the RP-HPLC-purified P-pyridoxyl peptide obtained from NaB[3H]4-reduced mouse ODC by digestion with endoproteinase Lys-C. The sequence and radioactivity of the 20 first amino- terminal residues of the single 3H-labeled peptide isolated by RP- HPLC from a Lys-C digest of NaB[3H]4-reduced ODC (Fig. 1) were determined with the Applied Biosystems 477A Protein Sequencer, as described under "Experimental Procedures." An X denotes a cycle for which no PTH derivative could be identified.

to proteolytic attack by endoproteinase Lys-C by virtue of its NaB[3H]4-mediated pyridoxylation.

Isolation of Sites of Binding of DFMO to Mouse ODC- Initial experiments showed that after inactivation by reaction with [5-I4C]DFMO, 19.1 nmol of the inhibitor was incorpo- rated per mg of purified ODC after gel filtration and extensive dialysis, corresponding to 19.7 nmol of enzyme subunit (based on an M , of 50,730). Thus, the stoichiometry of DFMO binding to recombinant mouse ODC is very close to 1 mol/ subunit as determined previously for wild-type mouse ODC (28, 29).

The adduct formed between DFMO and mammalian ODC has been reported to be remarkably stable to treatments such as heating in the presence of HC10, or sodium dodecyl sulfate, precipitation with (NH,),SO,, or extensive dialysis (28). In the present experiments no detectable loss of radioactivity could be measured after extensive dialysis of recombinant mouse ODC labeled with [5-14C]DFM0 even in the presence of 8 M urea, and prior treatment of the adduct with NaBH, or NaCNBH, had no effect on the stability of the label under these conditions. However, exposure to low pH under dena- turing conditions as used for peptide isolation resulted in a considerable loss of radioactivity from [5-'4C]DFMO-labeled ODC (data not shown). Thus, the DFMO-ODC adduct peptide(s) may be considerably more stable than the complexes formed in the reactions of other PLP-dependent enzymes with mechanism-based inhibitors, such as the irreversible inactivation of alanine racemase (43) and histidine decarboxylase (36,44) by 2-chloroalanine and 2-fluoromethylhistidine, respectively. These adducts were very labile upon denaturation of the protein and were found to be greatly stabilized by its reduction with NaBH,. Despite the apparently stable nature of the DFMO-ODC adduct, we included a NaBH, reduction step prior to denaturation of the enzyme to ensure stability of the labeled peptides during isolation and to minimize the reactivity of possible condensing groups in the DFMO-derived moiety of the adduct (23).

RP-HPLC analysis of the reduced [5-I4C]DFMO-inactivated ODC digested with endoproteinase Lys-C indicated the presence of two radioactively labeled peptides (peptides 24 and 29) (Fig. 3). Most (82-92% depending on the digest) of the total radioactivity associated with these two peptides was recovered in peptide 29. The latter peptide was sufficiently pure for direct automated sequencing, which was performed for 28 cycles. The 24 amino acid residues which could thus be identified unequivocally were identical with the predicted sequence extending from Tyr-350 to Val-377 in wild-type mouse ODC (4-6). Radioactivity in the eluent was first detected at the 11th cycle during the automated Edman degradation of fraction 29, which did not release any identifiable PTH-derivative (Fig. 4). This position corresponds to Cys- 360 in the mouse ODC sequence.

The predicted Lys-C peptide containing Cys-360 should include 69 residues. Since it was not possible to sequence this peptide completely and the recovery of radioactivity during the automated Edman degradation is not quantitative, the possibility that other I4C-labeled residues were present in peptide 29 in addition to Cys-360 could not be ruled out. Therefore, a portion of peptide 29 was digested with endoproteinase Glu-C (37) and the peptides separated by RP-HPLC. Virtually all (299%) of the radioactivity was associated with a single peptide (Fig. 5). The sequence of the first 18 amino acids of this peptide and the position of the labeled residue in this fragment were found to be identical to the corresponding amino-terminal residues in the undigested peptide 29 (results not shown). These data and the amino acid composition of

154 Structure of Mouse Ornithine Decarboxylase

0.4

I 0'3 v

0.2 v) r <

0.1

0.0

"---IT Peptide 29

10 i 20

1 eptide 2

a p , ~ . m , o I c 30 40 50

% Acetonitrile FIG. 3. RP-HPLC analysis of [5-"C]DFMO-labeled, NaBH4-

reduced ODC holoenzyme after digestion with endoproteinase Lys-C. Purified recombinant mouse ODC (18 nmol) was inactivated with 360 nmol of [5-''C]DFMO, reduced with NaBH4, carboxymethylated, and digested with Lys-C as described under "Experimental Procedures." The amount injected was 63 fig. The position of undigested ODC is shown by an arrow. Other details are as given in Fig. 1 and under "Results."

Cycle number

FIG. 4. Partial amino acid sequence and site of labeling of RP-HPLC-purified peptide 29 obtained from digestion of ["C] DFMO-labeled mouse ODC by endoproteinase Lys-C. The sequence and radioactivity of the first 28 amino-terminal residues of peptide 29 isolated by RP-HPLC from a Lys-C digest of [5-14C] DFMO-labeled ODC (Fig. 3 ) were determined as described in Fig. 2. An X denotes a cycle for which no PTH derivative could be identified.

this peptide (data not shown) clearly indicate that the Glu-C fragment that was sequenced is the peptide extending from Tyr-350 to Glu-368 in the native mouse ODC sequence and establish Cys-360 as being the only ["CIDFMO-labeled amino acid residue in peptide 29. (The two very minor peaks of radioactivity associated with slightly earlier retention times in the Glu-C digest may have resulted from partial cleavage by the endoproteinase a t Asp-361 and/or Asp-364. The reason for the very low yield of proteolytic cuts at aspartic acid residues by the Glu-C enzyme observed in the present case using a buffer in which both glutamic acid and aspartic acid residues should be sites for proteolytic cleavage (37) is unclear, but steric hindrance by the adduct present at Cys-360 could have limited proteolysis in the adjacent region of the peptide.)

The second ["CC]DFMO-labeled peptide identified in the Lys-C digest (peptide 24) was purified further by a second passage on RP-HPLC, and the sequence of its first 19 amino acid residues was determined (Fig. 6). A single PTH-derivative was detected a t each degradation cycle, except at the 12th cycle, in which no residue could be identified and radioactivity started to be eluted. The sequence was found to be identical to that determined for the P-pyridoxyl-containing peptide

6 - 0.15 5 : -

I 4 E

V - - 3s

2 z

t

.a > z -

L

0

1 r . 0

0.00 0 3-

20 30 40 50 60 % Acetonitrile

FIG. 5. RP-HPLC analysis of the peptides generated by digestion of [5-'4C]DFMO-labeled peptide 29 with endoproteinase Glu-C. Peptide 29 was purified by RP-HPLC from a Lys-C digest of [5-"C]DFMO-labeled mouse ODC as illustrated in Fig. 3 and digested with endoproteinase Glu-C as described under "Experi- mental Procedures." The positions of undigested ODC and peptide 29 in this chromatographic system are shown by arrows. Other details are as given in Fig. 1.

0 2 4 6 8 10 12 1 4 16 18 20

Cycle number

FIG. 6. Partial amino acid sequence and site of labeling of RP-HPLC-purified peptide 24 obtained from digestion of ['"C] DFMO-labeled mouse ODC by endoproteinase Lys-C. The sequence and radioactivity of the 19 first amino-terminal residues of [5-'4C]DFMO-labeled peptide 24 (cf. Fig. 3 ) were determined as described in Fig. 2. An X denotes a cycle for which no PTH derivative could be identified.

(Fig. 2), including the position of the unknown labeled residue, which corresponds to Lys-69. A greater relative amount of radioactivity was released at the cycles immediately after cycle 12 (Fig. 6) than was observed with the ["HIP-pyridoxyl peptide (Fig. 2), as well as with the other DFMO-derived adduct (Figs. 5 and 7). There was also a marked drop in the yield of amino acid residues observed starting a t cycle 13. These findings suggest an especially poor efficiency of cleavage of the DFMO-modified lysine residue. These results indicate clearly that in addition to Cys-360, catalytically activated DFMO binds to Lys-69 (the PLP binding site) in about 10% of reacting ODC subunits.

The exact relative proportions of the DFMO adducts at Lys-69 and Cys-360 are not certain because the digestion of the enzyme by Lys-C was never complete, and it is possible that one of the peptides is produced more readily than the other. However, analysis of the tryptic peptides described below confirmed that no more than 15% of the adducts occur at Lys-69.

Structural Characterization of the Cys-360-bound Adduc- To obtain a sufficient quantity of a peptide containing the DFMO-Cys-360 adduct for analysis by FAB-MS, the DFMO- inactivated recombinant mouse ODC (and a small fraction inactivated with [I4C]DFMO to act as a labeled marker) was


1 0 2 0 3 0 4 0 5 0 6 0

% Acetonltrile

FIG. 7. RP-HPLC analysis of the tryptic peptides obtained from [5-’4C]DFMO-labeled. NaBH,-reduced mouse ODC. Ap- proximately 400 pg of [5-”C]DFMO-labeled, NaBH4-reduced and carboxymethylated mouse ODC was digested with 10 pg of HPLC- purified bovine trypsin. An amount equivalent to 220 pg of the tryptic digest was then analyzed by RP-HPLC using a reversed-phase column (Bio-Rad RC-318) as described under “Experimental Procedures.” The absorbance range was extended to 1.0 units at 25.8 min. The positions of the four radioactive peptides detected (a, b, c, and d ) are indicated.

100

( A ) ’” 60

4 0

20 301

1903.6

l l

I l i

lO0l 16) 18!

90-1

80-

70-

60-

30

20

10

0 1890 1895

I 190

.7

FIG. 8. FAB mass spectra of two peptides isolated from the trypsin digest of DFMO-bound NaBH4-reduced mouse ODC. The region of the protonated molecular ion (M + H)’ is shown in each case for A, peptide a, and B, peptide b as designated in Fig. 7 .

digested to completion with trypsin. The resulting peptides were separated by RP-HPLC on a Bio-Rad Hi-Pore RC-318 column, which gives excellent resolution of small peptides. This separation showed the presence of four radioactive peaks a-d (Fig. 7). Peptides a and b, which, as shown below, corre- sponded to two forms of the Cys-360-containing peptide that together accounted for more than 80% of the total adducts, were analyzed by FAB-MS. The region surrounding the protonated molecular ion of each tryptic fragment is shown in Fig. 8, A and B, respectively. Although only a limited mass range is displayed, no other significant products were detected

in either sample. The spectrum in Fig. 8A shows an m/z value of 1,903.6 for the most intense peak in the (M + H)’ isotope cluster for tryptic fragment a. The nominal molecular mass indicated by the isotope distribution is 1,901 or 83 daltons higher than the 1,818 molecular mass of the proposed tryptic fragment YYSSSIWGPTCDGLDR. The protonated molecular cluster in Fig. 8B shows an m/z 1,899.7 as the most intense peak and indicates that the minor tryptic fragment b had a nominal molecular weight of 1,897. Amino acid analysis run on peptides a and b yielded almost identical results: Tyr (1.71, 1.77), Ser (2.77, 2.66), Gly (1.90, 2.061, Pro (0.87, 0.94), Thr (1.22,0.90), Cys (0.97, 1.30), Asx (2.34,2.42), Arg (0.97,0.91); the values in parentheses correspond to a and b, respectively. The presence of a large sample-related peak which eluted in the vicinity of leucine and isoleucine restricted quantitation of these residues. In addition, tryptophan could not be quan- tified reliably because of degradation commonly associated with this residue during hydrolysis. Nonetheless, the results confirm that both peptides a and b were derived from the proposed tryptic fragment containing Cys-360.

The results obtained by FAB-MS enabled the nature of the covalent adduct to Cys-360 to be deduced. As shown by the data in Fig. 8, the molecular mass increase caused by the adduct in Cys-360 was 83 and 79 daltons, respectively for peptides a and b. Both of these values are less than the molecular weight of either DFMO or PLP. Assuming that inactivation was caused by the alkylation of cysteine, the number of plausible elemental compositions for each adduct is relatively few. A further constraint is that both adducts must contain an odd number of nitrogen atoms since the molecular weights were increased by an odd number (nitrogen rule). The most likely composition for the alkylating group of the major product (peptide a ) is C5HI,,N. Based on the data obtained from FAB-MS, amino acid analysis, and peptide sequencing, it is apparent that DFMO binds to Cys-360 to give an S-((2-(l-pyrroline))methyl)cysteine derivative which is oxidized to the S-((2-pyrrole)methyl)cysteine adduct (mi- nor) or reduced to S-((2-pyrrolidine)methyl)cysteine in the presence of NaBH, (major). The structures for the observed adducts to peptides a and b are shown in Fig. 9 and are consistent with the proposed model for the action of DFMO (24). In the original mechanism for inactivation of ODC by DFMO (22), the exact nucleophile on the protein which binds to DFMO was not specified. The sequencing data in Fig. 4 indicate that the PTH-derivative resulting from position 360 of mouse ODC could not be identified and contained the bulk of the ’“C radioactive label. These observations point strongly to the thiol on Cys-360 as the nucleophilic group. Further, these data eliminate other potential nucleophilic residues on the peptide sequenced (e.g. tyrosine, serine, threonine, argi- nine) as binding sites for DFMO. Unfortunately, the amino acid analysis data acquired for peptides a and b could not be used to corroborate these findings since the covalent adducts to these peptides were apparently cleaved under the conditions used for hydrolysis. The amino acid analysis data did confirm, however, that peptides a and b were products of the same tryptic fragment which contained Cys-360.

Spectral Characteristics of Native and DFMO-inactivated Mouse ODC-The visible spectrum of purified recombinant mouse ODC exhibits two absorption maxima, namely at 339 and 418 nm (Fig. 10). The presence of two absorption maxima close to these two wavelengths is characteristic of a-decarboxylases (39, 45, 46). Several possible enzyme-bound coenzyme forms, including an uncharged Schiff base (enolimine form), an internal aldimine, or pyridoxamine 5”phosphate (45, 47, 48), can account for the maximum a t 330-340 nm.

156 Structure of Mouse Ornithine Decarboxylase

DFMO.

I

FIG. 9. Mechanism proposed for 7”- the inactivation of mouse ODC by F-

4 , +

s-cys=@ J

(minor)

[ DFMO + NaBH,

_j L 280320%0400440W520 280320360400440480520 28032036040044048052

Wavelength, nm

FIG. 10. Spectrophotometric characterization of native and DFMO-inactivated recombinant mouse ODC. A, time course of the spectral changes exhibited by purified recombinant mouse ODC holoenzyme (5 p~ in buffer A, pH 7.5) after the addition of 100 ~ L M DFMO. The various curves were obtained at 23 “C at the times (in min) indicated. B, comparison of the holo- and apoenzyme forms of native and DFMO-inactivated recombinant mouse ODC. Incubation of ODC with DFMO was carried out for 1 h at 37 “C before spectra were taken. ODC apoenzyme was prepared as described under “Ex- perimental Procedures.” Results for the holo- and apoenzyme forms were obtained using an enzyme concentration of 5.0 and 3.7 p ~ , respectively, at 23 “C in buffer A (pH 7.5). C, effect of NaBH, reduction on the absorption characteristics of DFMO-inactivated mouse ODC holoenzyme. The spectra were obtained at 22 “C before and after a 2-h incubation of the purified enzyme (100 p ~ ) with 1 mg/ml NaBH, in buffer A.

The peak at 410-430 nm characteristically reflects the absorption of the charged aldimine (ketoenamine form) between PLP and the lysine residue on the enzyme. The pure enzyme has also an absorbance maximum a t 277-278 nm; an extinction coefficient value of 10.1 was calculated at X = 278 nm (0.1% ODC (w/v) in buffer A after NaBH, reduction; results not shown). Upon addition of a 20-fold molar excess of DFMO, the peak at 418 nm decreased progressively while the absorbance at 339 nm dropped suddenly in the first 3 min after the addition of the inhibitor and then slowly underwent a secondary increase thereafter (Fig. 10A).The A339/A418 ratio increased approximately 1.8-fold (from 1.3-1.6 to 2.3-3.0) after complete enzyme inactivation by DFMO, although the

(major) PY ?-Lysm@

PY = H& ““)y OPO, - H

final A 3 3 9 value was lower than that found in unreacted ODC (Fig. 10, A and B ) . An inverse relationship between the absorbances at these two maxima has been described for the PLP-dependent histidine decarboxylase from Morganella morganii upon its inactivation by 2-fluoromethylhistidine (36, 44). However, in the latter case there was no initial decrease of the absorbance at the lower wavelength. As found with bacterial ODCs (16, 49), recombinant mouse ODC holoenzyme was readily (160 min) resolved by the addition of L- cysteine in a phosphate buffer, and prior inactivation with DFMO showed no protection in that respect (Fig. 10B). Reduction of DFMO-inactivated mouse ODC with NaBH, entailed the complete disappearance of the absorption maximum at 418 nm and a shift of the absorbance maximum from 339 to 331 nm (Fig. lOC), reflecting mostly the reduction of an internal pyridoxylidene imine between Lys-69 and PLP. An identical change was observed upon NaBH, reduction of the native holoenzyme (data not shown). The spectrum of the NaBH4-reduced enzyme was unaffected by dialysis as well as by treatment with L-cysteine in phosphate buffer (data not shown). Taken together, these results are consistent with PLP being bound to the DFMO-inactivated enzyme in the form of an uncharged Schiff base or an internal aldimine which can be easily cleaved out by the competing formation of the thiazolidine between L-cysteine and PLP.

DISCUSSION

Although a detailed understanding of the active site of ODC will require knowledge of the three-dimensional structure, the present results provide considerable information on key re- gions of the protein. The Lys-69 found to bind PLP is found in all known eukaryotic ODC sequences and is contained within a heptapeptide, -PFYAVKC-, which is conserved in all of these ODCs (4-13),’ suggesting that it is an essential part of the active site. The sequence differs completely from that of the peptide forming the cofactor binding site of the E. coli ODCs, which is -QSVHKQ- (14, 15). There is also no similarity with the apparent consensus sequence for PLP binding sites of several decarboxylases and other PLP-dependent enzymes, which is -SXHK- (21). It has been sug- gested that the histidine in this sequence helps to bind the PLP by forming an ionic interaction with the phosphate group

Struc ture of Mouse Ornithine Decarboxylase 157

(21 and references therein). Such an interaction could not occur in ODC, and this may provide an explanation for the finding that ODC is very readily resolved from its PLP cofactor (50). However, ODC is by no means unique in lacking this histidine residue since at least 10 other PLP-dependent enzymes do not have it (21), including mammalian histidine decarboxylase (51).

It is also noteworthy that mammalian ODC has long been known to be extremely dependent on the addition of high concentrations of thiol-reducing agents such as dithiothreitol for the maintenance of activity (50). This requirement pre- sumably relates to the importance of keeping critical cysteine thiol groups in the reduced form. The cysteine residues present at position 70 (adjacent to the PLP-lysine) and position 360, which is the major DFMO binding site, are strong can- didates for this requirement. The presence of two such residues in the vicinity of the active site may explain the extraor- dinary dependence of ODC on reducing agents. This requirement is in sharp contrast to bacterial histidine decarboxylase which is inhibited by dithiothreitol by virtue of an interaction with the bound PLP cofactor (39).

The Cys-360 binding site is contained in a nonapeptide -WGPTCDGL(I)D-, which is present in all known eukaryotic ODC sequences (4-13).2 This conservation provides addi- tional evidence that this residue is likely to be located close to the active site of the enzyme. The two conserved peptides surrounding the amino acids found to bind DFMO (residues 64-70 containing the PLP binding site and residues 356-364) are among the longest contiguous sequences conserved in the various eukaryotic ODCs. Two other relatively conserved sequences are residues 164-171 and 193-201. Mutation of amino acids within these sequences, namely Lys-169 to Ala and His-197 to Ala, abolishes all activity, indicating that these sequences are also likely to contribute toward the catalytic site (18).

The stoichiometry of the reaction of DFMO with ODC and the good recovery of the labeled peptides allow us to be confident that we have accounted for all of the binding of this inhibitor which is responsible for the inactivation of the enzyme. The scheme in Fig. 9 depicts the mechanism proposed for the irreversible inhibition of ODC by DFMO. DFMO is accepted by the active site of ODC where it forms a Schiff base to PLP. Subsequent decarboxylation of DFMO followed by elimination of a fluoride anion generates a conjugated imine. This reactive electrophilic imine is capable of alkylating the nucleophilic thiol group of Cys-360. Subsequent elimination of another fluoride anion yields a second conjugated imine, which then undergoes a transaldimination reaction with the amino group of Lys-60. The corresponding enamine formed by this reaction may then cyclize internally with a concomitant loss of ammonia to give a cyclic imine, S-( (2-( 1- pyrro1ine))methyl)cysteine. This structure is consistent with the spectroscopic evidence obtained and represents the major species formed by alkylation of ODC by DFMO.

There is only a minor covalent interaction of the reactive species formed from DFMO with the PLP-binding lysine of ODC. This contrasts with studies of other PLP-dependent enzymes in which a major binding site of enzyme-activated irreversible inhibitors is this lysyl residue (52-54). Such binding can occur via the nucleophilic attack of the lysine on a highly electrophilic imine metabolite of the inhibitor, but for several enzymes it has been shown that the inactivation occurs via a more complex pathway first described by Metzler and colleagues (52) in which the activated form of the inhibitor engages in a nucleophilic attack on the aldimine carbon of the cofactor to initiate the normal transaldimination process

leading to product release and reformation of the lysine-PLP Schiff base. The liberated reactive enamine intermediate can then react with the reformed internal aldimine. The reactivity of this intermediate can lead to labeling of the PLP-binding lysine or to reaction with other residues in the vicinity of the active site (52-56). In the present experiments, the spectral studies and the stability of the DFMO-ODC adducts suggest strongly that the covalent adducts do not retain the PLP. Therefore, the former pathway appears more likely to be responsible for the formation of both adducts. As described above, such an imine addition reaction is entirely consistent with the formation and structure of the cysteine adduct. However, the lysine adduct on ODC, which we have not yet been able to characterize, represents such a small fraction of the total binding that it remains possible that this is formed via the Metzler enamine pathway.

The observation that the major inactivating adduct formed between DFMO and ODC is formed by a nucleophilic attack of a cysteine residue on the conjugated imine is strikingly different from the mechanism of inactivation of histidine decarboxylase by monofluoromethylhistidine (36,44). In this case, the mechanism of inactivation clearly does proceed via a form of the Metzler enamine pathway. Although a single serine residue subsequently becomes labeled as a result of the covalent interaction with the inhibitor coenzyme derivative, this adduct was only isolated after reduction. Mutation of this serine to an alanine residue does not prevent the inactivation of histidine decarboxylase (44). Inhibition is therefore brought about by the tight binding of the inhibitor coenzyme complex and possibly by reaction at multiple sites. Another example of multiple sites of binding is the inactivation of ornithine aminotransferase by 4-aminohex-5-ynoate (55). Several products were found including reaction at Lys-292 (the PLP binding site) and Cys-388. The former adduct also contained the coenzyme, and convincing evidence that the enamine pathway is responsible for this inactivation was obtained.

It is apparent from these considerations that the mechanism of inactivation may depend on both the structure of the inhibitor and the residues available in the protein sequence in close proximity to the active site. The presence of a reactive cysteine to act as a nucleophile and the strong electrophilicity of the imine formed from DFMO seem to be sufficient to inactivate ODC primarily through the formation of the cysteine adduct. Many other enzyme-activated inhibitors of ODC have now been synthesized (23-25), and it is possible that this mechanism may not apply to all of them. This possibility is consistent with the studies on the inactivation of alanine racemase by fluoroalanine derivatives (54), in which inactivation by monofluoroalanine proceeds via the Metzler enamine pathway but with trifluoroalanine via direct covalent attachment of the electrophilic intermediate as the result of a nucleophilic attack by the amino group of the lysine residue. The partition ratio of this inactivation by trifluoroalanine was about 3 (54), which is similar to that for the inactivation of ODC by DFMO (29).

Acknowledgments-We are indebted to Anne Stanley for her expert assistance in the peptide sequencing and amino acid analysis, to K. E. Cornelius for amino acid analysis of the tryptic fragments, and to Dr. R. H. Davis for the information on the derived amino acid sequence for N. crassa ODC cDNA.

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