Stimulation ofGlucose Catabolism inEscherichia coli bya Potential Futile Cycle
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synthase (Pps) (EC2.7.9.2) inEscherichia coli stimulated oxygen consumption inglucose minimal medium. A further increase inPpsoverexpression to30-fold stimulated glucose consumption byapproximately 2-fold andresulted inanincreased excretion ofpyruvate and acetate. Insertion oftwocodons atthePvuII site intheppsgeneabolished theenzymatic activity andeliminated theabove-described effects. Boththeactive andtheinactive proteins weredetected atthepredicted molecular weight bypolyacrylamide gelelectrophoresis. Therefore, theobserved physiological changes wereduetothe activity ofPps.Thehigher specific rates ofconsumption ofoxygen andglucose indicate apotential futile cycle between phosphoenolpyruvate (PEP) andpyruvate. Amodelforthestimulation ofglucose uptake ispresented; itinvolves anincreased PEP/pyruvate ratio caused bytheoverexpressed Ppsactivity, leading toastimulation ofthePEP:sugar phosphotransferase system.Keywords:
Catabolism
PEP group translocation
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Chick-embryocells,transformed with Roussarcomavirus, showenhancedratesofsugartrans- portand glycolysis. Determination ofintracellular con- centrations ofglycolytic intermediates suggests thatthe enhancedglycolytic fluxisdueto-increased activities of hexokinase (ATP:D-hexose 6-phosphotransferase- EC 2.7.- 1.1), phosphofructokinase, (ATP:D-fructose-l-phosphate 6-phosphotransferase, EC 2.7.1.56), and pyruvatekinase (ATP:pyruvate 2-0-phosphotransferase, f:C2.7.1.40), and notdirectly totheincreased glucose transport. Thiscon- clusion issupported bythefinding thattheintracellular concentration offreeglucose isdecreased, ratherthanin- creased, inthetransformed cells. The presentobserva- tionssuggest thattheincreased glycolytic fluxisrelated toan increased rateofphosphorylation ofglucose, and thathexokinase inthetransformed cells isatleast partly released fromitsnormalcontrolmechanisminvolving feedback inhibition byglucose-6-P. A marked increase intherateofsugaruptakehasbeenre- ported forchick-embryo cells transformed byRoussarcoma virus(1-11). Thisincreased transport ofglucose isanearly event intransformation (8)andmayberelated totheincreased glycolytic fluxthathasbeenreported intransformed cells (3,12-17). Inorder todetermine therelationship between this increased glycolytic flux andtheenhanced sugartrans- port, andtoidentify themetabolic step(s) affected bytrans- formation, wehaveexamined thechanges inactivities ofkey glycolytic enzymesandalsothechanges inthelevels of glycolytic intermediates. Thestudies on enzymelevels, reported elsewhere (18), haveestablished thattheactivities ofhexokinase, phosphofructokinase, andpyruvate kinase, therate-limiting enzymes ofglycolysis, areincreased several- foldsoonafter infection ofchick embryocells withthe Schmidt-Ruppin strain ofRoussarcoma virus. Thatthese changes areassociated withtransformation rather thanwith virus infection wasestablished byexperiments witha tem- perature-sensitive mutant(Ts-68); infection withthis mutant atthenon-permissive temperatures, wherethecells arenot transformed, didnotaffect thelevels oftherate-limiting enzymes (18). Inorder toconfirm thatthese increases inenzymeactivities areindeed related totheincreased glycolytic fluxinvivo, wehavenowexamined thechanges inintracellular levels of glycolytic intermediates. Theincreases anddecreases inthe levels ofthese intermediates determine thecrossover points, whicharethemostreliable indicators oftheinvivosites ofmetabolic regulation (19). Thestudies reported heredemon- strate thattheincreased glycolytic flux invirus-transformed cells isrelated totheincreased intracellular activities of hexokinase (ATP:D-hexose 6-phosphotransferase, EC2.7. 1.1) phosphofructokinase
Hexokinase
Phosphotransferases
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Glucose inethanol-glycerol mixtures inhibits growth ofSaccharomyces cerevisiae mutants lacking phosphoglycerate mutase. Asuppressor mutation thatrelieved glucose inhibition wasisolated. Thismutation, DGTI-1 (decreasing glucose transport), wasdominant andproduced pleiotropic effects eveninanotherwise wild-type background. GrowthoftheDGTI-1mutant inglucose wasdependent onrespiration, andnoethanol was detected inthemediumwithin 7hofglucose addition. Whengrownonglucose, themutanthadareduced glucose uptake andboththelow-andhigh-affinity transport systems wereaffected. Ingalactose-grown cells, only thehigh-affinity glucose transport system wasdetected. Thissystem hadsimilar kinetic characteristics in thewildtypeandinthemutant. Catabolite repression ofseveral enzymes wasabsent inthemutantduring growth inglucose butnotduring growth ingalactose. Incontrast withthewildype, themutantgrownin glucose hadhightranscription oftheglucose transporter geneSNF3andnotranscription ofBXTJandBIT3. Expression ofmulticopy plasmids carrying theIXTI,HXT2,orHXT3geneallowed partial recovery ofboth fermentative capacity andcatabolite repression inthemutant. Theresults suggest thatDGTIcodesfora regulator oftheexpression ofglucose transport genes. Theyalso suggest thatglucose flux might determine the levels ofmolecules implicated assignals incatabolite repression. Addition ofglucose orother easily fermentable sugars to cultures ofSaccharomyces cerevisiae produces repression ofthe transcription ofmanygenes (16, 22,33), inactivation ofseveral enzymes, andactivation ofothers like phosphofructokinase, pyruvate kinase, andplasma membrane ATPase(for areview, seereference 15). Inspite oftheimportance ofthis general catabolic control exerted byglucose, itisnotknownhowthe yeast cell senses thepresence ofglucose andtransduces this information totrigger allthementioned phenomena. The important question oftheexistence ofaglucose receptor remains unanswered, inspite ofclaims that theproduct ofthe TPS1gene(coding fortrehalose-6-phosphate synthase [2, 37]) isthegeneral glucose sensor (35). Inorder toidentify elements implicated inthesensing andtransduction ofthe signal forglucose presence, wehavestarted theisolation of mutants withaltered responses toglucose. Ourstrategy wasto search forsuppressors ofthetoxic effect ofglucose inamutant defective inphosphoglycerate mutase. Thisenzyme, codedfor bytheGPM1gene(23), catalyzes theinterconversion between 3-and2-phosphoglycerates (14) andistherefore anessential enzymeforglucose metabolism. Glucose doesnotallow growth ofmutants affected indifferent steps oftheglycolytic pathway andinhibits their growthon alternative carbon sources (7,8).We reasoned thatamongsuppressors ofthe inhibitory effect ofglucose ongpmlmutants, wecould uncover mutants affected inthesensing andtransducing machinery of glucose, intransport orphosphorylation ofthis sugar, orin catabolite repression. We usedayeast strain withachromosomaldeletion oftheGPM1geneasstarting material. We report inthisarticle thecharacterization ofa dominant suppressor mutation, DGTJ-1, thatpresents animportant
Mutase
PEP group translocation
Snf3
Phosphoglycerate mutase
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