Abstract
Two enzymatic activities, 2-acylglycerolphosphoethanolamine (2-acyl-GPE) acyltransferase and acyl-acyl carrier protein (acyl-ACP) synthetase, were solubilized and purified from Escherichia coli membranes. Electrophoretic analysis of the final product of the purification procedure revealed a single protein species with an apparent molecular mass of 27 kilodaltons. The ratio of acyltransferase to synthetase activities remained the same throughout the purification scheme indicating that both activities were catalyzed by the same enzyme. 2-Acyl-GPE acyltransferase exhibited an apparent ACP Km of 64 nM under standard assay conditions that increased to 10 microM when the assay was conducted in the presence of 0.4 M LiCl. Acyl-ACP synthetase activity was not detected in the absence of 0.4 M LiCl, and the apparent ACP Km for this reaction was 16 microM. Direct evidence that ACP was a subunit of the acyltransferase/synthetase was obtained by the adsorption of both catalytic activities to an ACP-Sepharose affinity column and by the binding of [3H]ACP to the purified enzyme preparation. The apparent Km for acyl-ACP was 13 microM, and the rate of acyl transfer from this acyl donor was enhanced by the addition of 0.4 M LiCl indicating that the exchange of enzyme-bound ACP for acyl-ACP was a determinant factor in the rate of phosphatidylethanolamine formation from acyl-ACP. These data indicate that the 2-acyl-GPE acyltransferase and acyl-ACP synthetase reactions are catalyzed by the same membrane protein that possesses a high affinity binding site for soluble ACP.
Highlights
Acyl-ACP was 13KM,and therate of acyl transfer from acyltransferase is reminiscent of the properties of acyl-ACP. This acyl donorwas enhanced by the additionof 0.4M LiCl indicating that the exchange of enzyme-bound ACP for acyl-ACP was a determinant factor in thraete of phosphatidylethanolamineformationfromacylACP
The apparent K, for ACP in the standard 2-acyl-GPE acyltransferase assay was 64 nM indicating high affinity binding of ACP to the acyltransferase (Fig. 4). This high affinity activation of 2-acyl-GPE acyltransferase was abolished by the presence of 0.4 M LiCl in the assay (Fig. 4)
The addition of 0.4 M LiCl stimulated PtdEtn together, these results provide strong support for the concluformation from [14C]acyl-ACP,whereas LiCl was inhibitory sion that 2-acyl-GPE acyltransferase and acyl-ACP synthewhen ACP, ATP, MgClz, and 14C-fattyacid were used
Summary
Twoenzymaticactivities, 2-acylglycerolphospho- remaining in the membrane is phosphorylated by diacylglycethanolamine (2-acyl-GPE) acyltransferase and acyl- erol kinase to yield phosphatidic acid which re-enters acyl carrier protein (acyl-ACP) synthetase, were sol- the mainstream of the E. coli phospholipid biosynthetic pathbdttsbsuihcpyabrvheaeliitltenctoihipmiezeneeusesses.rds.iwiarfEnTeimaicdlmhtneaehiedctacipetriaaornnuatnoneztirpinidypaofhgmrtipohoeopetdrcefhfea.eartard2soicetc-uamynbAmrltoeatcertnmryhaealnov-tlahGsylecEefrsaPetcoislisrEuoeuvcafldhgisaattheericheroyeimstuaclotthwarfssiasiieatsnnnyhrasgneoecfplfltoeehcrplr2oaepiaudt7trsaaruomeilskfcyteeieiteazclmixooceanf----dtionbiawramneccaacneyycyytetllae-acr(bArtl3eimiooaC)dl.lneiPtcmAolaridcepblelyaorPyrlcapitatnvmdriceaooEtottnieietivvsneniebentzt,iysyheoamsa2rn(t4-adeaa)tcoct.ttrcytThiahcvlinhu-esaGsretpf1ePderr-sorEeupscsnroaueiosfncslaintysgttieiltnoistytsgrnhtaearenr2oecisfl-fimafadiPeetcsaerdtytadedulsif-ErtetaGahotttne(Pti4y’ortE-hn6afaer)irc.oseoitmfdtTrhasathleanhsiinnnseoshibited an apparentACP K , of 64 nM under standard assay conditions that increased to 10PM when the assay the presence of ATP and Mg2f [5,6].Acyltransferase activity is completely inhibited by monospecific ACP antibodies inwas conducted in thepresence of 0.4M LiCl. 2-Acyl-GPE acyltransferase and acyl-ACP synthetase activities were measured using the standard assay conditions described under “ExDerimental Procedures.’’ Kinetic Characteristicsof the Purified AcyltransferaselSynthetase-As reported previously [5,9],both 2-acyl-GPE acyltransferase and acyl-ACP synthetase exhibited an absolute requirement for ATP and M C , and the apparent K, for palmitic acid was 8 p~ for both enzyme reactions.
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