DMFO J. Biol. Chem. 1992 Poulin 150 8
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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 USA. Mechanism of the Irreversible Inactivation of Mouse Ornithine Decarboxylase by a-Difluoromethylornithine CHARACTERIZATION OF SEQUENCESATTHEINHIBITOR AND COENZYME BINDINGSITES* (Received for publication, August 12,1991) Richard PoulinSg, Li LuS, Bradley Ackermannll, PhilippeBeyll, 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 en- zyme used for determination of the binding site for pyridoxal 5”phosphate and of the residues modified in the inactivation of the enzyme by theenzyme-acti- vated irreversible inhibitor, a-difluoromethylorni- thine (DFMO). Thepyridoxal5”phosphatebinding lysine in mouse ODC was identified as lysine 69 of the mouse sequence by reduction of the purified holoen- zyme form with NaB[“HI4followed by digestion of the carboxymethylated protein with endoproteinase Lys- C, radioactive peptide mapping using reversed-phase high pressure liquid chromatography and gas-phase peptidesequencing. Thislysine 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 ad- ducts 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 knowneu- karyote ODCs. These results provide strong evidence that these two peptides form essential parts of the catalytic siteof 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 oxi- dized to S-((2-pyrrole)methyl)cysteine or converted to S-((2-pyrrolidine)methyl)cysteine by NaBH4 reduc- tion. 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. Proteinsequencing 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 thereforebe 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 inhib- itor. 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 biochem- ical 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 se- quences 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) andthe 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 mam- malian proteins and an81% (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 isavailable concerning the amino acids forming the active site of ODC. Complete loss of enzymatic activity of the mouse ODC occurs with the muta- tion 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 consensussequence 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 by guest, on October 25, 2009 www.jbc.org Downloaded from
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Mouse ornithine decarboxylase (ODC) was expressedin Escherichia coli and the purified recombinant enzymeused for determination of the binding site forpyridoxal 5”phosphate and of the residues modifiedin the inactivation of the enzyme by the enzyme-activatedirreversible inhibitor, a-difluoromethylornithine(DFMO). The pyridoxal 5”phosphate bindinglysine in mouse ODC was identified as lysine 69 of themouse sequence by reduction of the purified holoenzymeform with NaB[“HI4 followed by digestion of thecarboxymethylated protein with endoproteinase Lys-C, radioactive peptide mapping using reversed-phasehigh pressure liquid chromatography and gas-phasepeptide sequencing. This lysine is contained in thesequence PFYAVKC, which is found in all knownODCs from eukaryotes. The preceding amino acids donot conformt o theco nsensus sequenceo f SXHK, whichcontains the pyridoxal 5”phosphate binding lysine ina number of other decarboxylases includinOgD Cs fromE. coli. Using a similar procedure to analyze ODClabeled by reaction with [5-’*C]DFMO, it was foundthat lysine 69 and cysteine 360 formed covalent adductswith the inhibitor. Cysteine3 60, which was themajor adduct accounting for about 90% of the totallabeling, is contained within the sequence-WGPTCDGL(I)D-, which is present in all known eukaryoteODCs. These results provide strong evidencethat these two peptides form essential parts of thecatalytic siteo f ODC. Analysis by fast atombo mbardment-mass spectrometryo f tryptic peptides containingthe DFMO-cysteine adduct indicated that the adductformed in then zyme was probably thcey clic imineS -((2-(l-pyrroline))methyl)cysteine. This is readily oxidizedto S-((2-pyrrole)methyl)cysteine or converted toS-((2-pyrrolidine)methyl)cysteine by NaBH4 reduction.This adduct is consistent with spectral evidenceshowing that inactivation of the enzyme with DFMOdoes note ntail the formatioonf a stable adduct between the pyridoxal 5’-phosphate, the enzyme, and the inhibitor.
Transcript of DMFO J. Biol. Chem. 1992 Poulin 150 8
THE~OLI RNAI . OF BIOLOCICAI.CHEMISTRY ( 1 1992 hy The American Societyfor BiochemistryandMolerular B101oc.y. I nc. Vol. 267, No.I ,Issue of J anuary 5, pp.150-158,1999 Printed~n U S A .Mechanism of theIrreversibleInactivationof Mouse Ornithine Decarboxylase by a-Difluoromethylornithine CHARACTERIZATION OFSEQUENCES AT THE I NHI BI TORANDCOENZYMEBINDINGSITES* (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 CollegeofMedicine, Hershey, Pennsylvania I7033 and the TMarion Merrell Dow Reseaich Institute,Cincinnati, Ohiob52I5 Mouse ornithinedecarboxylase(ODC) wasexpressed in Escherichiacoliandthepurifiedrecombinanten- zyme usedfor determinationofthebindingsitefor pyridoxal5phosphateandoftheresiduesmodified intheinactivation ofthe enzymebytheenzyme-acti- vated irreversible inhibitor, a-difluoromethylorni- thine(DFMO). Thepyridoxal5phosphatebinding lysinein mouse ODC wasidentifiedas lysine 69 of the mouse sequencebyreductionofthe purifiedholoen- zymeform with NaB[HI4 followed bydigestion of the carboxymethylatedproteinwithendoproteinaseLys- C, radioactivepeptidemappingusingreversed-phase highpressure liquid chromatography and gas-phase peptidesequencing.Thislysineis containedin the sequencePFYAVKC,whichisfoundinallknown ODCs fromeukaryotes.Theprecedingaminoacidsdo notconformtotheconsensussequenceof SXHK, which containsthepyridoxal5phosphatebindinglysinein a number of otherdecarboxylasesincludingODCs from E.coli.Usinga similar procedure to analyzeODC labeledbyreactionwith[5-*C]DFMO, itwasfound that lysine 69 andcysteine 360 formedcovalentad- ductswiththeinhibitor.Cysteine360, which wasthe major adduct accounting for about90% ofthetotal labeling,iscontainedwithinthesequence -WGPTCDGL(I)D-, which is presentinallknowneu- karyote ODCs. Theseresultsprovidestrong evidence that these two peptides form essential partsofthe catalyticsiteofODC. Analysisbyfastatombombard- ment-massspectrometryof trypticpeptidescontaining the DFMO-cysteineadductindicatedthattheadduct formedintheenzyme wasprobablythecyclicimineS- ((2-(l-pyrroline))methyl)cysteine. This is readily oxi- dizedto S-((2-pyrrole)methyl)cysteineor convertedto S-((2-pyrrolidine)methyl)cysteine byNaBH4reduc- tion. Thisadduct is consistentwithspectralevidence showingthatinactivation of the enzyme with DFMO doesnotentailtheformationof a stableadductbetween * This researchwassupportedinpart byGrants CA-18138 and CA-37606 from the National Institutes of Health.Proteinsequencing was made possible by National Science Foundation Biological Facility CenterGrant DIR 8804758. The costs of publicationof this article were defrayed in part by the paymentofpage charges. This article musttherefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Supportedbya postdoctoralfellowshipfromtheMedicalRe- search Council of Canada. Present address: MRC Group in Molecular Endocrinology,Lava1 UniversityMedicalCenter,Quebec, Canada GIV 4G2. 11 To whom correspondenceshould be addressed Dept. of Cellular and Molecular Physiology and Pharmacology, P. 0. Box 850, Hershey Medical Center,PennsylvaniaState University College of Medicine, Hershey, PA17033. thepyridoxal5-phosphate, the enzyme, andtheinhib- itor. L-Ornithine decarboxylase (ODC) is animportant enzyme which,in mammaliancellsand many othereukaryotes,is essential for the productionofputrescine,thediaminepre- cursor of the polyamines. Theactivityof ODC is very rapidly andgreatlychangedinresponsetostimuliaffectingcell growth and polyamine content,andtheunderlying biochem- ical mechanisms responsible for thesechanges havebeen the subjectofextensive investigation(1-3).The structure and enzymaticproperties of ODC have received muchless atten- tion although recent studies inwhichthe cDNAhasbeen clonedhaveallowedthe derivationofthe aminoacidse- quencesforODCs from a number of sourcesincluding mouse (4-6), human (7), rat(8,9),hamster(lo),Xenopus laeuis (ll), Trypanosomabrucei(12), Saccharomycescerevisiae(13),and Neurosporacrassa.? The eukaryoticODCsequencesshowverylittle, ifany, similarityto thoseofthe Escherichiacolibiosynthetic and biodegradativeODCs(14-16)and theLactobacillussp.30a ODC(17). However,comparisonsoftheeukaryoticODC sequences show aremarkablesimilarity between ODCs from these sourceswithmorethan 90% identity between themam- malian proteinsandan81% (Xenopus),69% (Trypanosoma), 42%(Neurospora), and 40%(yeast) identitybetweenthese proteinsandthemurine ODC over the common core region of the enzyme. Littleinformationis availableconcerning the amino acidsforming the active site of ODC. Complete loss of enzymatic activity of the mouse ODC occurswith themuta- tion to alanineofeither His-197orLys-169(18), and an inactivehamster ODC mutant was foundto be caused by a change of glycine toaspartic acid at position 381 (19). Trun- cation of the mouse ODC atthe carboxyl endto remove the terminal 37amino acids did not affect the activity(18, 20). Even thesiteof binding of the pyridoxal 5phosphate(PLP) cofactor was not known, and eukaryotic ODC does notcontain theconsensussequence of -SXHK- which includes thePLP- binding lysinein many PLP-dependentenzymes 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 pressureliquidchromatography;FAB, fast atombom- 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.crassaODC (personal communication). 150 by guest, on October 25, 2009www.jbc.orgDownloaded from Structure ofMouseOrnithineDecarboxylase151 DFMO was designedas an enzyme-activatedirreversible inhibitorofODC(22-24).Aspredicted,incubationofthe eukaryote enzyme withDFMO leads toanirreversible loss of enzymeactivity. Theinhibition ofT. brucei ODC is ofmajor pharmacologicalimportance sinceAfrican sleepingsickness caused bythis organismisveryeffectivelytreated bythis drug, which is now in clinical usage for this purpose. DFMO mayalso have therapeutic potentialfor anumberof other illnessescausedbyprotozoansandfordiseasesinvolving abnormal cellular proliferation, including cancer (25-27).As predicted from the proposedmechanism (22-24),theinacti- vation of mouse ODC by DFMO involves the decarboxylation of DFMO by the enzyme andthestoichiometricbindingof a metabolite to the protein(28, 29).In the present paperwehavedeterminedtheaminoacidresiduesin mouse ODC which are involvedinthecovalentbindingofDFMO, determined thestructureof the major adduct,andlocated the lysyl residue forming a Schiff base with PLP. Theseresults provide addi- tionalinformationonthemechanismof action of DFMOand ontheactive site of ODC. EXPERIMENTALPROCEDURES Materiak-~-[l-~C]Ornithine(52 Ci/mol)and NaB[H], (490Ci/ mol)wereobtainedfromDuPont-NewEnglandNuclear.[5-I4C] DFMO (60 Ci/mol) was obtained from the AmershamCorp. Unla- beledDFMOwas producedbythe Marion-MerrellDowResearch Institute (Cincinnati, OH).N-phosphopyridoxyl-N-tert-butyloxy- carbonyllysine,synthesized as described(30), wasgenerouslypro- vided by Dr. s. F. Yang (University of California,Davis). RP-HPLC- purifiedtrypsinfrombovinepancreas(sequencinggrade,essen- tially(
