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THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1993 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 268, No. 33, Issue of November 25, pp. 24572-24579,1993 Printed in U. S. A .

Effect of Mutations at Active Site Residues on the Activity of Ornithine Decarboxylase and Its Inhibition by Active Site-directed Irreversible Inhibitors*

(Received for publication, April 23, 1993, and in revised form, July 8, 1993)

Catherine S. Coleman$, Bruce A. Stanley, and Anthony E. PeggO From the Departments of Cellular and Molecular Physiology and of Pharmacology, The Milton S. Hershey Medical Center, Pennsylvania State University College of Medicine, Hershey, Pennsylvania 17033

Mouse ornithine decarboxylase (ODC) and mutants changing residues thought to be involved at the active site were expressed in Escherichia coli, purified to homogeneity by affinity chromatography on a pyridoxamine 5’-phosphate-agarose affinity column, and tested for their kinetic properties and their inactivation by enzyme-activated irreversible inhibitors. All of the mutant enzymes were expressed at comparable levels to the wild type protein (2-4% of the total soluble protein), all bound to the affinity column, and there were only small differences in the apparent K , values for L-ornithine providing strong evidence that the mutations did not lead to any gross changes in the protein structure. The mutation K69A led to a change in the spectrum of the enzyme and a 550-fold decrease in the k,,JK, (specificity constant) value. These results are consistent with lysine 69 being the residue that forms a Schiff base with the pyridoxal 5’-phosphate co-fac- tor. Mutation C70S did not greatly affect the activity despite its proximity to this lysine but increased the K , about 2-fold. In contrast, the mutation C360A greatly reduced the specificity constant (by 26-fold) despite a 2-fold decrease in the K,, suggesting that this cysteine residue is critically involved at the active site. Although cysteine 360 is known to be the major site of binding of the inhibitor, a-difluoromethylornithine (DFMO), the C360A mutant was still sensitive to inhibition by this drug. However, the kinetics of inactivation were altered, the partition ratio was 10 times greater, and the labeled adduct formed by reaction with [5-14C]DFM0 was removed from the protein under some denaturing conditions. This adduct was found to occur at lysine 69. The K69A mutant was also sensitive to DFMO with a lower partition ratio than the wild type enzyme. These results indicate that inactivation of ODC by DFMO can occur via interaction with either of two separate residues that form essential parts of the active site. This renders it unlikely that resistant mutants will arise from changes in the enzyme structure. In contrast to the results with DFMO,

* This research was supported by National Institutes of Health Grant CA-18138. Protein sequencing was made possible by an Na- tional Science Foundation Biological Facility Center Grant DIR 8804758. Some equipment was provided by a grant from the Alcoa Foundation. 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.

$ The grateful recipient of a Wellcome Trust Travel Grant. To whom correspondence should be addressed Dept. of Cellular

and Molecular Physiology, The Milton S. Hershey Medical Center, Pennsylvania State University College of Medicine, P.O. Box 850, Hershey, PA 17033. Tel.: 717-531-8152; Fax: 717-531-5157.

the C360A mutant ODC was completely resistant to inactivation by (R,R)-b-methyl-a-acetylenicputrescine and was much less sensitive than the wild type enzyme to a-monofluoromethyldehydromethylornithine, showing that the reactive species formed from these inhibitors either cannot be formed by this mutant or are unable to react with lysine 69. Finally, the well known, extreme reliance of mammalian ODC on the presence of thiol-reducing agents to maintain activity is probably explained by the critical role of cysteine 360, since the C360A mutant was much less sensitive to inactivation by incubation in the absence of dithiothreitol.

L-Ornithine decarboxylase (ODC)’ is an important enzyme that is necessary for the biosynthesis of polyamines in mammalian cells and in many microorganisms (1-4). ODC activity is essential for a normal cell growth rate unless exogenous polyamines are provided. The inhibition of ODC activity interferes with the growth of many cells including parasitic protozoa, and ODC is a valuable target for the design of therapeutically useful compounds (5, 6). One such inhibitor, a-difluoromethylornithine (DFMO) is an excellent anti-try- panosomal agent that is now in clinical use for the treatment of African sleeping sickness caused by Trypanosoma brucei brucei. Numerous other inhibitors of ODC have been synthesized, some of which may represent improvements over DFMO in terms of potency and pharmacokinetic parameters, but their clinical utility remains to be demonstrated (6-8).

Despite extensive investigation of the physiological regu- lation of ODC activity and the effects of ODC inhibition described above, there is only a modest amount of information available on the structural and catalytic features of the enzyme. The primary structure of ODC from 10 different eukar- yotes has been derived from cDNAs (6, 9) and confirmed by sequencing peptides derived from proteolytic cleavage of the recombinant mouse enzyme (10). ODC is dependent on pyridoxal 5”phosphate (PLP) for activity, and the eukaryotic ODCs consist of dimers of identical subunits of M , 49,000- 77,000 (6, 11). Preliminary results obtained using the mouse ODC suggest that the PLP binding site is at lysine 69, and this lysine and several surrounding residues are conserved in all the known ODC sequences (10). The major site of binding

The abbreviations used are: ODC, ornithine decarboxylase (EC 4.1.1.17); DFMO, D,L-a-difluoromethylornithine; PLP, pyridoxal 5’- phosphate; MAP, (R)-a-ethynyl-(R)-6-methylputrescine also known as (R,R)-6-methyl-a-acetylenicputrescine; PAGE, polyacrylamide gel electrophoresis under denaturing conditions; HPLC, high pressure liquid chromatography; RP, reversed-phase; A-MFMO; cu-monofluoromethyldehydromethylornithine.

24572

StructurelActivity of Ornithine Decarboxylase 24573

of DFMO was found to be at cysteine 360 (lo), and this amino acid is contained in a conserved pentapeptide -GPTCD- (6). This peptide may, therefore, form part of the active site.

Mammalian ODC is known to be very sensitive to oxidation, and full activity in in vitro assays requires the addition of sulfhydryl reducing agents such as dithiothreitol (1, 12, 13). This suggests that there are key cysteine residues in the molecule, and one of these may be cysteine 360 described above. Another candidate cysteine residue is cysteine 70, which is adjacent to the putative PLP-binding lysine 69.

In order to investigate the amino acids forming the active site of ODC in more detail, we have carried out site-directed mutagenesis of lysine 69, cysteine 70, and cysteine 360 in mouse ODC and expressed the mutant enzymes in Escherichia coli. The purified enzymes were then used for investigation of their spectral and kinetic properties, their reliance on thiol- reducing agents, and the response to enzyme-activated irreversible inhibitors such as DFMO and MAP ((R,R)-&methyl- a-acetylenicputrescine).

EXPERIMENTAL PROCEDURES

Materials-Oligodeoxynucleotides were synthesized in the Macro- molecular Core Facility, Hershey Medical Center, by using a Milligen 7500 DNA synthesizer. [c~-~~SlThio-dATP, ~-[~~S]methionine, and L- (1-"Clornithine were purchased from DuPont NEN. [1-14C]DFM0 (18.3 Ci/mol) and [5-14C]DFM0 (60 Ci/mol) were obtained from the Amersham Corp. Unlabeled DFMO and all other enzyme-activated irreversible inhibitors of ODC were generously provided by the Mar- ion Merrell Dow Research Institute (Cincinnati, OH). Plasmid pGEMSzf(-), helper bacteriophage M13K07, and RNasin were obtained from the Promega Corporation (Madison, WI). In vitro transcription and rabbit reticulocyte translation systems were purchased as kits from Ambion Inc. (Austin, TX). Sequenase 2.0 and guanidine hydrochloride (Ultrapure) were obtained from U. S. Biochemical Corp. Restriction enzymes and DH5aMCR E. coli cells were obtained from Bethesda Research Laboratories. T4 DNA Ligase was purchased from New England Biolabs (Beverly, MA). Proteinase K and purified trypsin from bovine pancreas (sequencing grade) were obtained from Boehringer Mannheim. Ampicillin, dithiothreitol, isopropyl P-D-thiogalactopyranoside, kanamycin, pyridoxamine 5'-phosphate, PLP, NaCNBH3, CF,COOH, and iodoacetate were purchased from Sigma. Brij 35 (30% (w/v) solution) was obtained from Technicon Instru- ments Corporation (Tarrytown, NY). NaBH4 was supplied by Aldrich (Milwaukee, WI). The E. coli strain EWH331 (14) was a generous gift from Drs. C. W. and H. Tabor (NIDDK, Bethesda, MD). Anti- serum against mouse ODC was raised in rabbits (15, 16).

Site-directed Mutagenesis of Active Site Residues of Mouse ODC- The plasmid pGEM-ODC-ER, which contains the mouse cDNA with an EcoRI restriction site at positions 22-26 relative to the A of the initiation codon (10) was used to make site-directed mutants of ODC. Oligodeoxynucleotide-directed mutagenesis was carried out as described (17) using a modification of the method described by Kunkel (18). Single-stranded DNA containing uracil was prepared from pGEM-ODC-ER grown in the E. coli strain CJ236 in the presence of M13K07 helper bacteriophage and purified on Qiagen-Tip 100 anion exchange columns (Qiagen Inc., Chatsworth, CA). The orientation of the f' origin of replication in pGEM-ODC-ER was such that the single-stranded DNA produced contained the DNA equivalent to sense mRNA, so the following antisense oligodeoxynucleotides were synthesized for use as mutagenic primers to produce the indicated amino acid changes (mismatches are underlined) as follows: K69A,

AGACTTGACT-GCG-3'; C360A, 5"CAAGGCCATCAGCTGTTG GEC-3 ' ; C360S, 5'-CCGATCAAGGCCATCAaTGTTGGTCCC (2-3'.

The double-stranded DNA formed in the mutagenesis reaction was introduced into the E. coli strain DH5crMCR by electroporation (19). Ampicillin-resistant colonies were picked, grown up overnight, and used to prepare DNA (20). This double-stranded DNA was sequenced using Sequenase and a suitable primer to identify the plasmids containing the desired mutation. Plasmid DNA from the wild type pGEM-ODC-ER and derived mutants were prepared from 500-ml cultures using alkaline lysis and purification on Qiagen-Tip 500 anion exchange columns.

5"GCTATCGTTACACEGACTGCG-3'; C70S, 5"GCTATCGTT

Transcription and Translation of mRNA for Wild Type and Active Site Mutant ODC-Aliquots (50 pg) of plasmid DNA prepared from pGEM-ODC-ER and mutants derived from it were linearized with XbaI. Portions (10 pg) of linearized DNA were treated with 10 pg of Proteinase K for 30 min at 50 "C, and 1 pg was transcribed in vitro using T7 RNA polymerase (Ambion). The uncapped RNA prepared was used for the synthesis of ODC protein using a cell-free in vitro translation system (17). The RNA was heated for 10 min at 65 "C and cooled on ice immediately prior to use. Translation assays contained an uncapped master mix lacking methionine, [35S]methionine, and a nuclease-treated rabbit reticulocyte lysate (Ambion). After incubation for 90 min at 30 "C, 25-p1 aliquots were separated by SDS- PAGE and the labeled bands corresponding to ODC quantified by scanning the gels with a Betascope 603 Blot Analyzer. Translation assays to determine ODC activity were carried out after synthesis of the protein in lysates as described above except for the substitution of unlabeled methionine for the labeled amino acid. The products from 50-p1 translation reactions were then assayed for ODC activity (16, 17).

Subcloning of Wild Type and Mutant Mouse ODC into a Protein Expression Vector-The cDNAs encoding wild type and mutant ODCs were subcloned into the pIN-III-lppP'5-A3 (21) protein expression vector as follows. The wild type and mutant ODC inserts were isolated from pGEM-ODC-ER constructs by digestion of purified plasmid with EcoRI and EamHI. The 1.6-kilobase fragments were purified after agarose gel electrophoresis using Geneclean@ (BIO 101, La Jolla, CA). These fragments were ligated into the PIN-111 protein expression vector, which wacsolated after digestion of the PIN-ODC- 5 construct (10) with EcoRI and EamHI and gel purified as described above. The ligation products were used to transform E. coli EWH331, a strain that does not contain any endogenous ODC activity because of a mutation in the spe C gene (14). All transformants were sequenced to confirm the presence of the desired mutations.

Expression of Wild Type and Active Site Mutant Mouse ODC- EWH331 cells transformed with the pIN-III-lppP~5-A3 vector containing wild type or active site mutant cDNAs were grown in a modified M9 medium. This contained 50 pg/ml ampicillin, 0.75% (w/v) Casa- mino acids (Difco, Detroit MI), 2.4% (w/v) D-glucose, 0.1 mM CaCl,, 1 mM MgSO,, 1 mM thiamine HC1, and M9 salts (42 mM Na2HP04, 22 mM KH,P04, 19 mM NH4C1, and 8.5 mM NaCl). Culture conditions were as follows. Ampicillin-resistant colonies from frozen stocks streaked onto M9 agar were inoculated into 10 ml of M9 medium and grown up overnight a t 37 "C (10). For small scale cultures, a 2.5-ml aliquot of each bacterial suspension was added to 25 ml of M9 medium in 125-ml conical flasks and grown at 37 "C with shaking at 225-250 rpm. Isopropyl P-D-thiogalactopyranoside (0.2 mM) was added to the cultures when the Am was between 0.6 and 0.8 and the cultures allowed to grow to an Asoo of 2.0-2.5. Cells were harvested in 35-ml Sarstedt centrifuge tubes. The pellets were then washed with buffer A (25 mM Tris-HC1, pH 7.5, 0.1 mM EDTA, 2.5 mM dithiothreitol, 0.02% (w/v) Brij 35), frozen in liquid N,, and stored at -70 "C until assayed for enzyme activity. All subsequent procedures were con- ducted at 0-4 "C; soluble extracts were prepared after resuspending the thawed pellets in buffer A. These cell suspensions were sonicated for 8 min in an ice water bath (lo), and the supernatant collected after centrifugation at 11,500 rpm for 20 min was used to determine ODC activity and for analysis of protein size by SDS-PAGE (22). In order to detect protein that reacted with anti-ODC polyclonal anti- body, the proteins separated by SDS-PAGE were electrotransferred to a nitrocellulose sheet (Schleicher & Schuell) in a Bio-Rad blotting apparatus overnight at 30 V and 4 "C. Western immunoblot analysis was then performed using the ECL method according to the manu- facturer's instructions (Amersham). Laser densitometry was used to determine the relative levels of ODC expression.

Purification of Wild Type and Mutant ODCs-Large scale cultures of wild type, C360A, C70S, and K69A ODCs were grown in the M9 medium defined above by inoculating 5 ml of a saturated culture/500 ml of medium in a 1-liter culture flask. These were cultured essentially as for the small scale cultures described above. The ODC purification steps were performed exactly as described by Poulin et al. (IO), the appropriate wild type or mutant ODC being eluted from a pyridoxamine 5'-phosphate-agarose affinity column with buffer A containing 50 p M PLP. The same fractions contained most of the ODC activity from the wild type and the mutant enzyme purifications. These fractions were pooled and concentrated using a Diaflo YM-10 membrane (Amicon, Beverly, MA) to a 3-5-ml final volume and the purity established by analysis of a portion of the purified protein on a 12.5% SDS-PAGE minigel using the Phast Gel system (Pharmacia).

24574 StructurelActivity of Ornithine Decarboxylase

Identification of the DFMO Binding Site in Recombinant Mouse C360A-During all the following operations, the purified enzyme solution was protected from light to reduce photo-decomposition of the cofactor and photo-oxidation of tryptophan residues. The concentrated purified enzymes obtained after affinity chromatography were supplemented with PLP to obtain a final concentration of 40 p~ of the coenzyme (as determined by its A41Z). Both wild type and C360A were allowed to react with a 20-fold molar excess of [5-14C]DFM0 for 1 h at 37 "C. Immediately following each inactivation, the DFMO- enzyme adduct was reduced with NaBH4 (I mg/ml) for 2 h a t room temperature as described by Hayashi et al. (23). Following dialysis against 1 liter of buffer A and 2 X 1 liters of 25 mM Tris-HC1, pH 8.5, 2 mM EDTA, 2.5 mM dithiothreitol, the enzyme was reduced and carboxymethylated under a N, barrier (10). Each carboxymethylated protein was dialyzed against 2 liters of water followed by 2 X 2 liters of trypsin digestion buffer (100 mM Tris-HC1, pH 8.5, containing 10% (v/v) HPLC grade acetonitrile). The volume was then reduced to 0.6- 0.8 ml using a Centricon-10 microconcentrator (Amicon) and digested with trypsin (1:20, protease:substrate ratio) for 18 h a t 37 "C in the presence of 1 mM CaC12. The tryptic peptides were analyzed by RP- HPLC with a Beckman HPLC system model llOA and a Bio-Rad Hi-Pore RP-318, 250 X 4.6-mm column using a linear gradient of 0- 62.5% acetonitrile over 60 min in the presence of 0.1% (v/v) CFaCOOH. The HPLC fractions containing the labeled peptides from the C360A digestion were collected manually into polypropylene Eppendorf tubes, dried down using a Speed Vac concentrator, and sequenced with an Applied Biosystems 477A Protein Sequencer. The radioactivity released at each degradation cycle was also determined.

Spectrophotometric Analysis of Wild Type and K69A ODC-Puri- fied wild type and K69A ODC (400 pg) in buffer A containing 50 WM PLP was applied to a PD 10 gel filtration column (Pharmacia) and eluted in PLP-free buffer A. Each holoenzyme was concentrated in a Centricon-10 a t 4 "C to a final concentration of 2 mg/ml. Buffer A was used as a blank for the spectrophotometric measurements, but the addition of 1 WM PLP to both enzyme and reference cuvettes did not alter the spectral characteristics observed in a parallel experi- ment. The spectral characteristics obtained at room temperature using a Beckman DU-65 spectrophotometer were determined between 280 and 540 nm prior to and following reduction with 2 mM NaCNBH3 at 4 "C for 12 h (24). This did not give complete reduction of the ODC preparation, and the spectra were also recorded after treatment with an additional 2 mM NaCNBH3 for 2 h at room temperature.

Determination of the Partition Ratio for Wild Type and Mutant ODC-Purified enzyme was incubated with 200 FM [l-"C]DFMO and 40 p~ PLP under conditions used for ODC assays for 2 h a t 37 "C. The I4CO, released was collected in hyamine hydroxide and determined as in ODC activity assays. The binding of [5-I4C]DFMO to ODC was determined in parallel incubations containing the same reactants; after incubation with 200 p~ [5-"C]DFMO for 2 h at 37 "C, the labeled enzyme was transferred to a Centricon-10 and washed free of unbound [5-14C]DFM0 with buffer A. The radioactivity re- tained by the Centricon membrane was determined by liquid scintil- lation counting. Bovine serum albumin and lysozyme were used as negative controls for nonspecific binding of DFMO, and the radioactivity present in these samples was subtracted from that present when ODC was used.

Miscellaneous Procedures-ODC activity was determined by measuring the release of I4CO, from ~-[l- '~C]ornithine during a 30-min assay a t 37 "C (16). One unit of ODC activity is defined as the amount of enzyme producing 1 @mol of "CO,/min. Protein was determined by the method of Bradford (25) using bovine serum albumin (fraction V; Miles Laboratories, Elkhart, IN) as standard.

RESULTS

Effect of Mutations on ODC Activity-As a rapid screen of the effects of mutations on ODC activity, a synthetic RNA was prepared from the pGEM plasmids containing the mu- tated cDNAs, translated in a reticulocyte lysate, and the resulting increase in ODC activity was measured. The synthesis of ODC protein was determined by a parallel incubation in the presence of [35S]methionine, and the activity results were corrected for differences in the amount of ODC protein synthesized. This was only a small correction, since the extent of synthesis was quite similar. The results showed that the mutations K69A, C360S, and C360A were totally inactive

within the limits of detection, which were about 2% of the control activity. The C70S mutation was active but had only one-quarter of the wild type activity.

These mutant cDNAs were then expressed in E. coli using the PIN expression vector system previously used to express the wild type mouse ODC (10). Expression was carried out in EWH331 cells, which have no endogenous ODC activity (14). The content of mouse ODC protein as measured on Western blots of the total soluble proteins in the E. coli extracts was not altered much by the mutations, amounting to 55, 99, 136, and 125% of the control value for the K69A, C70S, C360S, and C360A mutants, respectively. (The slightly lower value for the K69A mutant may be due to a more rapid degradation rate because of the inability to form a Schiff base with PLP as described below.) The ODC activity of the K69A and the C360S mutants in this system was <1 nmol/min/mg compared with 733 nmol/min/mg of the control ODC, confirming the virtual lack of activity of these mutants. However, the C360A mutant was clearly active (11 nmol/min/mg), albeit having less than 2% of the wild type activity. The C70S mutant had full activity when expressed in E. coli. This differs from the result found in the reticulocyte lysate above but is probably due to the higher K, for ornithine for this mutant ODC (see below), since the assays in the lysates were carried out with a low ornithine concentration.

Control, C70S, C360A, and K69A mutant ODCs were purified to homogeneity from E. coli extracts in a one-step procedure using affinity chromatography on a pyridoxamine 5"phosphate-agarose affinity column as previously described for wild type ODC (10). All of the mutant ODCs bound to this column and were eluted in a homogeneous form by the addition of PLP. Kinetic properties of these enzymes are shown in Table I. All of the mutant enzymes had some activity, but the K69A and C360A mutations had a great effect on the kcat values, decreasing them by 582- and 74-fold, respectively. The K , for L-ornithine of the K69A mutant was not altered, and the C360A mutant actually had a slightly lower K, for L-ornithine, leading to a specificity constant (kCat/K,) 26 times lower than the control. The C70S mutant had a higher K,,, than wild type ODC, but there was no change in the kcat (Table I).

Effect of C360A, C70S, and K69A Mutations on Inactivation by DFMO-Previous studies indicated that when ODC was inactivated by DFMO, an enzyme-activated irreversible inhibitor, the major adduct (amounting to at least 90% of the total) was formed a t cysteine 360 of ODC (10). It was therefore of particular interest to determine whether the C360A ODC mutant was still sensitive to DFMO. In fact, this mutant was inactivated by incubation with DFMO (Fig. l), and activity

TABLE I Comparison of properties of purified C360A, K69A, C70S and wild

type ODC Wild type and mutant ODCs were purified to homogeneity and K,

values determined from double-reciprocal plots of ornithine concentration and initial reaction velocity. The kcat values were determined as described by Fersht (46). Kinetic parameters for irreversible inactivation by DFMO were determined according to Kitz and Wilson (47). The partition ratio of 14C0, released from [1-14C]DFM0 to [5- I4C]DFMO bound to each enzyme was determined as described under "Experimental Procedures."

ODC used K, k,,,/K, K, DFMO ki..,tDFMO

p M M's" W min" Control 86 160,000 13 0.32 3.7 C360A 30 6,100 56 0.09 36.0 K69A 81 289 9.6 0.12 2.0 C70S 150 84,000 4.4 0.10 7.6


-1 00

C360A ODC

A

$- Non-mutant ODC 1 " " 1 , , ~ ~ 1 ~ ~ ~ ~ ~ " " ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ " , ,

0 10 20 30 40 50 60 Minutes

FIG. 1. Inactivation of ODC and C360A mutant ODC by DFMO. Results are shown for the inactivation of ODC (lowerpanel) and mutant C360A ODC (upper panel) when incubated with the concentration ( p ~ ) of DFMO indicated.

could not be restored by extensive dialysis (results not shown). However, the rate of irreversible inactivation of the C360A mutant was slower than for the control as indicated by a 3- fold decrease in the rate constant, kinact (Table I). This slower rate of inactivation is also reflected in the rate of production of 14C02 from [1-14C]DFM0 (Fig. 2).

It can also be seen from Fig. 2 that more 14C02 was released by the C360A mutant than from the wild type ODC or the K69A mutant. This indicates that the partition ratio between decarboxylation of the inhibitor and binding to enzyme is greater for the C360A mutant. This was confirmed by directly measuring this partition ratio using [5-'4C]DFM0 to inactivate the protein and [1-14C]DFM0 to measure the decarboxylation. These results showed a 10-fold increase from 3.7 for the wild type recombinant enzyme (this value is in good agreement with reported value of 3.3 with the mouse enzyme (26)) to 36 for the C360A mutant (Table I).

There was also a significant difference in the stability of the labeled adduct attached to ODC by reaction with [5-14C] DFMO. The label was completely stable to SDS-PAGE in the case of the control enzyme, as previously reported (15, 16). However, the [5-'4C]DFMO-labeled C360A mutant required reduction with NaBH4 for stability under these conditions (Fig. 3). Furthermore, most (77%) of the DFMO-labeled adduct could be recovered from the C360A mutant ODC by boiling for 5 min in 10% (v/v) acetonitrile (results not shown).

In order to determine the amino acid residue in the C360A mutant to which DFMO is bound, the protein was reacted with [5-14C]DFM0 followed by NaBH4, and the resulting labeled protein carboxymethylated and digested with trypsin. The resulting peptides were separated by reversed phase HPLC (Fig. 4). The great majority of the label from the wild type ODC was present in a peptide eluting at about 40 min that corresponds to the peptide YYSSSIWGTCDGLDR containing cysteine 360 as previously reported (10). The radioactivity from the C360A mutant ODC digest eluted in two peptides at 25 and 35 min. When subjected to sequencing in a gas phase sequencer, it was found that these peptides had

1 Non-mutant ODC - - 125 0 200

100

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- - 500

E " I 400 p' 100 P

K69A ODC h -

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0 10 20 30 40 50 60 Minutes

FIG. 2. Release of 14C02 from [l-'"C]DFMO by ODC, C360A, and K69A mutant ODCs. Each ODC preparation (2 pg) (upper panel, non-mutant; middle panel, C360A; and lower panel, K69A) was incubated with the concentration of [1-'4C]DFM0 ( p ~ ) indicated.

the sequence YAVXCNDSR and VTPFYAVXCNDSR, where the residue X contained the label and did not correspond to a known amino acid (Fig. 5 ) . This indicates that the trypsin digestion split part of the larger peptide at the internal phen- ylalanine residue to give rise to these two peaks, and subsequent experiments (not shown) with a lower trypsin:ODC ratio led to all of the label being recovered in the larger peptide. The residue X corresponds to lysine 69 in the ODC sequence. Therefore, it appears that the adduct inactivating the C360A mutant ODC is formed at lysine 69.

Both the C70S and the K69A mutants were readily inactivated by DFMO (Table I). The K69A mutant had a lower partition ratio of 2.0 than the wild type, which is consistent with the small amount of 14C02 formed from [1-14C]DFM0 by this protein (Fig. 2). The Ki of the K69A mutant was little changed from the wild type value, although the rate of inactivation by DFMO was slower. The C70S mutant also differed slightly from the wild type in the kinetics of inactivation, and, interestingly, it had a lower K, even though the K, for the ornithine substrate was greater (Table I).

Effect of C360A Mutation on Inactivation by Other ODC Inhibitors-There was an even more striking difference between the wild type and the C360A mutants in the inactivation by MAP (Fig. 6). In agreement with previous reports (7, 8, 27), MAP was a very potent inactivator of ODC, and more than 90% of the activity was lost after exposure to 0.1 PM MAP for 5 min. In contrast, the C360A mutant was not

24576 StructurelActiuity of Ornithine Decarboxylase

A B C D E

51 k D a *

FIG. 3. Effect of NaBH, reduction on stability of label in- corporated into ODC after reaction with [B-"CJDFMO. Soluble extracts from transformed EWH331 cells were prepared as described under "Experimental Procedures." 300 pg of wild type and C360A extracts and of bovine serum albumin were each incubated with 2.5 pCi of [5-I4C]DFMO (60 mCi/mmol) a t 37 "C for 30 min. Aliquots of these reactions were treated with either the equivalent volume of H 2 0 or NaBH, (6 mg/ml) for a further 2 h a t 37 "C. Reactions were boiled in the presence of SDS sample buffer for 3 min, and portions containing 65 pg of protein were analyzed by SDS-PACE. Lane A contained NaBH,-treated bovine serum albumin; lanes B and C show bands for wild type ODC treated without or with NaBH,, respectively; lanes D and E illustrate the absence and presence of [5-"C]DFMO bound to C360A ODC after boiling of the nonreduced and NaBH,- reduced enzyme, respectively.

affected significantly by incubation with 1 mM MAP for 60 min, indicating that the cysteine 360 residue plays a critical role in the interaction of MAP with ODC. The K69A mutant was shown to be sensitive to inhibition by MAP with 45% of the activity being lost after exposure to 1 PM MAP for 30 min (results not shown). The response of the wild type and C360A ODCs to inhibition by a-monofluoromethylornithine was similar to the findings with DFMO, but there was a marked difference in their sensitivities to its unsaturated analog, a- monofluoromethyldehydromethylornithine (A-MFMO) (8, 28). A-MFMO was a very potent inactivator of wild type ODC, producing a 53% reduction in activity within 15 min of exposure to 0.25 p ~ , but 50 PM A-MFMO was needed to decrease the C360A activity by a similar amount (52%) within 15 min (results not shown).

Effect of Q60A and C70S on Stability of ODC in the Absence of Thiol-reducing Agents-Wild type ODC and the C360A and C70S ODC mutants were stable when incubated in the presence of 2.5 mM dithiothreitol for 60 min at 37 "C. However, when incubated in the absence of this reducing agent, the wild type ODC was inactivated rapidly, losing 70% of its activity within the first 10 min and >90% within 60 min (Fig. 7). This is consistent with many earlier studies using crude extracts of mammalian cells in which it has been shown that the addition of high levels of dithiothreitol is necessary in order to get accurate estimations of ODC activity (12, 13). In contrast, the C360A mutant was quite stable in the absence of dithiothreitol losing <20% activity within 60 min. The C70S mutant showed an intermediate pattern. Over the first 20 min of incubation, it was quite stable and was similar to the C360A mutant, but it then lost activity quite rapidly over the next 40 min and was reduced to 10% activity by 60 min. These results suggest that the cysteine at position 360 is a particularly sensitive residue that must be maintained in a reduced state for the catalytic activity of ODC.

Effect of K69A Mutant on Spectra and PLP Binding-There was a significant difference between the absorption spectra of purified wild type and the PLP mutant K69A ODC over a

0.5

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t

0 10 20 30 40 50 60 70 80 minutes

FIG. 4. Separation of peptides derived by tryptic digestion from ODC and C360A mutant ODC inactivated by [S-"C] DFMO. Purified C360A and wild type ODC were inactivated with a 20-fold molar excess of [B-"C]DFMO, reduced with NaBH,, carboxymethylated, and digested with trypsin as described under "Experi- mental Procedures." Results shown are the RP-HPLC analyses of the peptides obtained after digestion of -350 pg of C360A ODC (panel A ) and wild type ODC (panel B ) with trypsin. The positions of the radioactive peptides (counts are from 2 0 4 aliquots of 15-ml fractions) from both trypsin-digested enzymes are shown in p a n e l C.

wavelength region between 280 and 540 nm (Fig. 8). In particular, the absorption maximum at 339 nm was absent in the K69A mutant. This can be attributed to several possible enzyme-bound forms including an uncharged Schiff base (en- olimine form) or an internal aldimine (29, 30). Both enzymes also showed a peak with a maximum at 415 nm, and this peak was decreased by reaction with 2 mM NaCNBHs for 12 h at 4 "C with a corresponding increase in the peak at 339 nm. Complete reduction of the 415 nm absorbing species of both wild type and K69A mutant enzymes required further treatment with 2 mM NaCNBHn for 2 h at room temperature (Fig. 8). This peak at 415 nm may be due to a charged aldimine (ketoenamine form) between PLP and other lysine residues in the ODC sequence.

DISCUSSION

The rapid test for the enzymatic activity of site-directed mutations in ODC set up by transcribing the plasmids containing ODC cDNAs with an RNA polymerase in uitro and then translating the resulting mRNA in a reticulocyte lysate (17) provides a convenient method for the preliminary screen of such mutations, but it should be noted that this assay is not sufficiently sensitive to detect the greatly reduced activity

StructurelActiuity of Ornithine Decarboxylase 24577

I h

v 8 300

250

200

1 50

100

50

0

R

V T P F Y A V ' C N D S R Amino Acid Released

from C360A ODC inactivated by [5-14C]DFM0. The enzyme FIG. 5. Location of radioactive adduct in tryptic peptides

was inactivated by reaction with [5-14C]DFM0, reduced with NaBH4, carboxymethylated, and digested with trypsin, and the labeled peptides were isolated as in Fig. 4. These peptides were then sequenced using an Applied Biosystems 477A gas phase protein sequencer, and the radioactivity released at each cycle is plotted. The amino acids released at each cycle are indicated along the abscissa in sequence. The first residue released from the smaller peptide (0) corresponded to the fifth residue from the larger peptide (0).

1 C360A ODC 1

t

I &

Non-mutant

A

ODC

Y

0 10 20 30 40 50 60

FIG. 6. Inactivation of ODC and C360A mutant ODC by MAP. Results are shown for the inactivation of ODC (lower panel) and mutant C360A ODC (upper panel) when incubated with the concentration (GM) of MAP indicated.

Minutes

of the C360A and K69A mutations. The expression of these cDNAs in E. coli and subsequent purification of the protein are needed to reveal these low activities. The substantial decrease in the specificity constant of ODC containing these mutations does provide strong evidence that cysteine 360 and lysine 69 are likely to be at the active site and play a major role in the enzymatic activity. This is consistent with the observation that inactivation of mouse ODC by [14C]DFM0 leads to the attachment of a radioactive adduct at these positions (10). Conclusions from experimental results obtained using enzymes altered by site-specific mutagenesis are always subject to some concern that the mutations may produce gross alterations in protein structure. Although this cannot be ruled absolutely for our work with ODC since no

0 10 20 30 45 60 Minutes

FIG. 7. Loss of ODC activity on incubation in the absence of dithiothreitol. The non-mutant ODC, the C70S ODC mutant, and the C360A mutant were each incubated in the absence of dithiothreitol for the time shown, and the activity remaining was determined.

W

C m

n 4

0.4

0.3

0.2

0.1

280 320 360 400 440 480 520 280 320 360 400 440 480 520

Wavelength (nm)

FIG. 8. Spectrum of non-mutant and K69A mutants. Absorb- ance spectra are shown for purified wild type ODC and the PLP mutant K69A ODC before and after reduction with NaCNBH3. The results were obtained at 23 'C using an enzyme concentration of 2 mg/ml in buffer A prepared as described under "Experimental Pro- cedures." Spectrum I shows the absorption characteristics of each enzyme prior to reduction. Spectrum 2 shows the spectral changes following reduction with 2 mM NaCNBH3 for 12 h at 4 "C. Spectrum 3 shows the effect on these spectra of treatment with an additional 2 mM NaCNBH3 for 2 h at 23 "C.

structures based on crystallography for either wild type or the mutants are yet available, it is unlikely, since the mutant enzyme cDNAs were expressed at similar levels to the wild type protein, and the mutant proteins were readily purified by affinity chromatography on pyridoxamine 5'-phosphate- agarose and bound PLP and L-ornithine relatively normally,

Our results using the K69A mutant are in agreement with the preliminary identification of this residue as part of the active site and forming a Schiff base with the PLP. The alterations in spectrum and dramatic loss of activity with a 500-fold reduction in the specificity constant are consistent with a major role for this residue. However, it is of some interest that the K69A ODC was not totally lacking in enzymatic activity. This contrasts to previous reports for the bacterial histidine decarboxylase (31) and with the work on several aminotransferases (24, 32, 33). This suggests that, in mammalian ODC, this lysine residue does not play an absolutely essential role in the catalytic mechanism. Roles that have been postulated for this residue include the following: allowing the substrate to form a Schiff base with the PLP via

24578 StructurelActiuity of Ornithine Decarboxylase

an internal transaldimination, aiding in the expulsion of the product by reformation of the complex with PLP, and providing the needed proton-withdrawing group. Although these functions are clearly not indispensable for activity, the lack of the lysine group to facilitate them may be responsible for the greatly reduced kcat value for the K69A mutant.

There must be a potential proton acceptor/donor in ODC, but this function is either not fulfilled by the PLP-binding lysine or else an alternative residue takes over this function in the mutant enzyme. There is precedence for the latter possibility. The mutation K145A of the PLP-binding-lysine in D-amino acid aminotransferase completely prevents the half-reaction generating pyruvate, whereas mutation K145R allows pyruvate formation to occur. When an external amine such as ethylamine was added, the K145A mutant was also able to bring about this reaction (24). Similarly, the K258A mutant of aspartate aminotransferase is inactive, but some activity can be restored by use of exogenous amines or by mutating residue 258 to arginine (33). Thus, in these trans- aminases, although the lysine normally takes part in the catalytic reaction, it can be replaced by another residue. Therefore, it is possible that this function in ODC is normally brought about by lysine 69, but, in the K69A mutant, it is taken over by another amino acid in the protein. However, in view of the significant activity of the mutant, the alternative hypothesis that some other amino acid always performs this function deserves consideration. Other candidates for the acidbase catalyst are cysteine 360, histidine 197, lysine 115, and lysine 169, all of which are needed for more than 2% activity (results presented here and Refs. 11 and 17).

The lack of a lysine at position 69 in ODC does not prevent the binding of PLP, since the K69A mutant enzyme was readily purified by affinity chromatography on pyridoxamine phosphate-agarose. It also does not affect the affinity for the substrate, L-ornithine, as indicated by the unaltered K, (Table I). These properties are similar to those of the K232A mutant histidine decarboxylase where alteration of the lysine forming a Schiff base with the P L P cofactor did not prevent substrate or PLP binding (31). Recently, Tsirka and Coffin0 (11) have reported that the mutation K69R greatly reduced the activity of mouse ODC and that the K69R mutant did not bind to a pyridoxamine phosphate Affi-Gel column. The binding of the ODC apo-enzyme to such a column obviously cannot involve a Schiff base formation with a lysine, since no aldehyde group is available. This report is in striking contrast to our results with the K69A mutant and suggests that the presence of an arginine residue at this position produces a significant distortion of the active site such that pyridoxamine phosphate is unable to bind.

The K69A ODC mutant was inactivated readily by incubation with DFMO and has a lower partition ratio than the native enzyme. This inactivation occurs by reaction of the conjugated imine, generated by decarboxylation followed by elimination of a fluoride ion, with cysteine 360 (8, 10). The lower partition ratio presumably results from a slower rate of elimination of the product from the active site of the enzyme that increases the chance of this electrophilic imine alkylating the nucleophilic thiol group of cysteine 360. The slower rate of elimination would occur because of the inability of the alanine at position 69 to react with the conjugated imine in the transaldimination that is normally accomplished by lysine 69.

The finding that C70S mutation does not greatly affect ODC activity is consistent with a recent report of the ODC sequence from Leishmania donouani showing that this enzyme (which has a much larger M, of 77,000) has a serine at the

equivalent position (34). The major changes with the mouse C70S ODC mutant were an increase in the K , and a change in the dependence on dithiothreitol. The L. donouani ODC was reported to have a K, of 200-500 FM, which is also much higher than for the control mouse ODC (35).

In contrast, the cysteine at position 360 is conserved in all known eukaryotic ODCs (6); mutation to alanine decreased the specificity constant by 26-fold, and mutation to serine was even more deleterious to activity; and this residue is the major site of interaction (-90%) when the mouse ODC is inactivated by DFMO (10). It is therefore clearly part of the active site. Several other PLP-dependent enzymes have been shown to have cysteine residues at the active site. These include cysteine 111 in 3,4-dihydroxyphenylalanine decarboxylase (36) and cysteine 298 in tryptophan indole-lyase (37). Also, cysteine 273 forms part of the L-ornithine binding site of ornithine transcarbamoylase (38). The extreme reliance of mammalian ODC on the presence of high levels of reducing agents such as dithiothreitol for optimal activity, which was first reported by Janne and Williams-Ashman (12), has been confirmed by many other studies, showing that ODC in crude tissue extracts can be reliably assayed only if these are prepared in the presence of such thiols (13). This property can largely be explained by the need to maintain cysteine 360 in the reduced form, since the C360A mutant was much less sensitive than the control ODC to lack of dithiothreitol (Fig. 7). The C70S mutant was slightly less sensitive than the wild type ODC, suggesting that the oxidation of this residue, which is adjacent to the P L P binding site, may also be deleterious.

Although the major site of binding of DFMO to control ODC was removed in the C360A mutant, this enzyme was still irreversibly inactivated by DFMO (Fig. 1). The partition ratio was substantially increased, and the rate constant for inactivation was reduced (Table I), but DFMO is still an effective and efficient inactivator of this enzyme. Inactivation by DFMO occurred by formation of an adduct at lysine 69 (Fig. 4). A small fraction (-40%) of the adducts formed when DFMO inactivates the wild type ODC also occurs at this site (lo), but this could not be shown, with certainty, to inactivate the enzyme, since it was such a small fraction of the total. The present results confirm that ODC can be irreversibly inactivated by virtue of a covalent modification of the PLP- binding lysine. This adduct is likely to be generated via the pathway first described by Metzler and colleagues (39) in which an enamine intermediate reacts with the internal aldimine between PLP and lysine. Such a reaction has been shown to be responsible for a number of inactivations of PLP requiring enzymes brought about by “enzyme-activated irreversible inhibitors” (8,39-43). We were unable to identify the nature of the adduct unequivocally owing to the absence of appropriate marker compounds, but its spectrum indicated the presence of the pyridoxal moiety and the fast atom bom- bardment mass spectrometry analysis of the tryptic peptide containing the adduct and of the adduct released from the protein by boiling in acetonitrile are consistent with this conclusion.’ The much greater partition ratio for the inactivation of ODC via this mechanism than via the direct alkyl- ation of cysteine 360 (10, 26) may be due to the increased reactivity of the enamine with solvent or to the efficient expulsion of the reactive species from the active site when cysteine 360 is not present. The higher partition ratio is consistent with the much smaller proportion of the lysine 69 adduct formed compared with the cysteine 360 adduct when the wild type enzyme is inactivated by DFMO (10).

* C. S. Coleman, B. Ackermann, and A. E. Pegg, unpublished observations.


Our results showing that both the K69A and C360A ODC mutants are readily inactivated by DFMO suggest that it is unlikely that resistance to DFMO via alterations in the enzyme structure will arise during prolonged treatment with this drug. Both residues that can be attacked to inactivate the protein are critical for activity. This is potentially important, since DFMO has potential as an anti-tumor agent and is already in use as an anti-trypanosoma1 agent (5-7). In both cases, quite large doses of DFMO are needed over a prolonged period and provide a strong selective pressure for the development of resistance. The major routes to DFMO resistance, at present, are ODC gene amplification in mammalian cells and in Leishmania and insensitivity to uptake or changes in S-adenosylmethionine metabolism in trypanosomatids (6, 44).

The lack of inactivation of the C360A mutant by MAP is very interesting, since MAP is one of the most potent inacti- vators of mammalian ODC and is believed to act in the same general way as DFMO by binding at the active site where proton addition via the microscopic reversibility principle generates the reactive species (8, 28, 45). Either this species cannot be formed by the enzyme or, more probably, it is unable to react with lysine 69. Similarly, the resistance of the C360A mutants to A-MFMO suggests that the reactive species generated at the active site is only poorly reactive with lysine 69. In any event, the use of these inhibitors, which has shown promise for several therapeutic strategies involving ODC inactivation (6), could be compromised by the development of resistant mutants.

Acknowledgments-We thank Anne Stanley for carrying out the peptide sequencing and Rukmani Viswanath for technical assistance.

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