1973•Unpublished venueRequires access
Kinetics andRegulation oftheSalt-Dependent Aspartate Transcarbamylase ofHalobacterium cutirubrum
Per Norberg, Joseph Kaplan, D. J. Kushner
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Abstract
Properties oftheaspartate transcarbamylase oftheextremly halophilic bacterium Halobacterium cutirubrum, an enzyme thatneeds highsalt concentrations foractivity andregulation, werestudied incell-free extracts. Theenzyme was stable on prolonged incubation at4C inconcentrated extracts (50mg of protein perml)butnotindiluted extracts. Mg2+ionsandA-mercaptoethanol stabilized enzyme activity. Atsalt concentrations below themaximumforactivity(3.5 M),theenzyme was rapidly inactivated. Carbamyl phosphate stabilized theenzyme undertheseconditions; aspartate hada smaller effect. Theenzyme was moststable at0C;raising orlowering thetemperature fromthis point increased therateofinactivation. Onexposuretolowered salt concentrations, enzyme activity was more sensitive thanfeedback inhibition. Hyperbolic substrate saturation curveswere foundforcarbamyl phosphate. TheKm obtained varied withthesalt concentration used. Withaspartate, sigmoidal curveswere foundwhenextracts were assayed immediately after preparation, buthyperbolic curveswere obtained withextracts allowed tostand1to2hr.Thepresenceofcytidine triphosphate (CTP)decreased theVmaxbutdidnotchange the Km;thisisthusa V-type enzyme.Lowconcentrations ofsuccinate activated theenzyme,inthepresenceandabsence ofCTP;higher concentrations didnot affect itsactivity. CTPincreased theactivation energyoftheenzyme in3.5 M salt butdecreased itin2.0 M salt. Atbothsaltconcentrations, thesensitivity oftheenzyme tofeedback inhibition diminished withincreasing temperatures. Gelchromatography suggested thattheenzyme incrudeextracts hada molecularweight of160,000. Precipitating theenzyme withpolyethylene glycol decreased themolecular weight to34,000, andthis activity was no longer sensitive toCTP.Thepresenceofeither substrate oftheenzyme during polyethylene glycol treatment prevented dissociation oftheenzyme andloss offeedback inhibition. Thus, as withother aspartate transcarbamylases, association ofsubunits seems toberequired forregulation ofactivity byendproduct.