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(