Proc. Nat. Acad. Sci. USA Vol. 72, No. 5, pp. 1802-1806, Mlay 1975
Reversible, Coenzyme-A-Mediated Inactivation of Biosynthetic Condensing Enzymes in Yeast: A Possible Regulatory Mechanism [a-isopropylmalate syntliase (2-isopropylmalate synthase)/homocitrate synthase/citrate (si)-synthase/ channeling of acetyl-coenzyme A]
JAMES W. TRACY AND GUNTER B. KOHLHAW* Department of Biochemistry, Purdue University, West Lafayette, Indiana 47907
Communicated by Herbert C. Brown, February 21, 1975 ABSTRACT a-Isopropylmalate synthase [3-hydroxy-4methyl-3-carboxyvalerate 2-oxo-3-mnethylbutyrate-lyase (CoA-acetylating); EC 4.1.3.12], the enzyme catalyzing the first committed step in leucine biosynthesis, and homocitrate synthase [3-hydroxy-3-carbox'-adipate 2-oxoglutarate-lyase (CoA-acetylating); EC 4.1.3.21], the first enzyme in lysine biosynthesis in yeast, are rapidly inactivated in the presence of low concentrations of coenzyme A, a product of both reactions. Closely related compounds like 3'dephospho-coenzyme A or oxidized coenzyme A are almost without effect, as are other sulfhys-dryl compounds. Citrate (si)-synthase [citrate oxaloacetate-lyase (pro-3S-CH2COO- - acetyl-CoA); EC 4.1.3.7] appears to be completely resistant against inactivation by coenzyme A. Ilnactivated a-isopropylmalate and lhomocitrate synthases can be reactivated by dialysis, but not by adding excess substrate. Protection against coenzyme-A-mediated inactivation is provided by relatively high concentrations of the a-ketoacid substrate or the specific end product inhibitor of each of the two enzymes. The coenzyme-A-mediated inactivation of a-isopropylmalate synthase has been more closely investigated. It requires the presence of divalent metal ions, with Zn ++ being most effective. The inactivation does not require molecular oxygen. It occurs in the presence of low concentrations of substrates and is observed in toluene-treated cells. These results, together with evidence that a-isopropy-lmalate syntlhase and lhomocitrate synthase are located in the mitochondria, suggest a mechanism by which increasing intra-mitochondrial coenzyme A concentrations might serve as a signal of decreasing acetyl-coenzyme A levels, triggering a temporary inactivation of biosynthetic acetyl-coetizyme A-consumnig reactions in order to channel the available acet-l-coenzy-me A into the citrate cycle.
inivestigate homocitrate and citrate synthase with respect to their CoA sensitivity. Homocitrate synthase [3-hydroxy-3carl)oxyadlipate 2-oxoglutarate-lvase (CoA-acetylating); EC 4.1.3.21] catalyzes the condensation of a-ketoglutarate and acetyl-CoA, thereby initiating the biosynthesis of lysine via the a-aminoadipate pathway. The enzyme is present in yeast (2). We report here that homocitrate synithase is also specificallv inactivated by CoA. By contrast, citrate synthase [citrate oxaloacetate-ly ase (pro-3S-CH2COO- acetyl-CoA); EIC 4.1.3.7] is not affected by CoA, even at concentrations far above those effective with the other enzymes. Results from exl)eriments designed to mimic in vivo conditions suggest that the (oA inactivation could occur in the living cell. Support for a possible physiological significance of the selective effect of (oA comes fromi evidence that both a-isopropyllmalate and homnocitrate svntlhase are located in the mitochondria (see
Discuission). A brief account of some of the results reported here has been
lresenite(l (3). M~NIATERIALS AND METHODS
Chemicals. CoA (90%lpure), oxidized CoA, 3'-dephospho(oA, andl (1,N6-etheno)-CoA were purchase(I from P & L lBiochemicals, Inc. The purity of CoA as supplied was confirmed by a stoichiomnetric assay using phosphotransacety'lase from Clostridiinm kliiyveri (4) an(l by titration of free sulfhydryl groups with 5,5'-ditlhiobis(2-niitrobenizoic acid) (Nbs2). a l)rep)ared by the quantitative acetylation of Acetyl-CoA -as (CoA with acetic anhvdride after the method of Simon and Shlemlin (5). Organismi. Comimercially obtained baker's yeast (3Budweiser ye\last, Anlheuser-Buschl, Inlc., St. Louis, MIo.) was used
The enzyme a-isolprol)plmalate svnthase [3-hvdroxv-42-oxo-3-metlhvlbutv rate-lv ase (CoA-acetylating); EC 4.1.3.12] catalyzes the first reaction in the leucine biosynthetic pathway, i.e., the formationl of aisoprol)lmalate from acetvl-CoA and a-ketoisovalerate via an aldol-condensation-tvpe reac tioli. P'revious work in this laboratory on the l)rol)erties of yeast ca-isol)ro)vllmalate synthase led to the recoolnitioni of a strong inactivating effect of free CoA which occurred wlien the enzyme watis incubated with CoA in the absence of other liganals or in the l)resence of acetyl-CoA (1). a-Ketoisovalerate and the feedback inhibitor, leucine, l)artially l)rotecte(l the enzyme against CoA inactivationl, but subsequent addition of substrates (lid not reverse it. The inactivating effect was sl)ecific for CoA and was not seen with other sulfhvd(lrvl or a(lenvlate comlpoundls.
methyl-3-carboxyvalerate
throuighout these studies.
Cell Disrtiption. (Crude extracts were lprepared by suspending 5 g of cells (wet weight) in 10 ml of 50 mMA potassium phosl)hate buffer, p)lI 7.5, containing 1.5 mM\ phenylmethylsulfonll fluoride an(l passing the susl)ension through a chilled (40) Aminliico French pressure cell at 20,000 pounds/inch2 (180 il)a). To thedisrultel suspension were added 20 ml of IpII 7.5 buffer containing 2.4 MI ammonium sulfate, 50 mM\ potassium l)hol)halte, and 1.5 mi \l phenylmethyl-sulfonl fluoride. After stirring for 30 min at 00, the mixture was centrifuged for 20 min at 30,000 X g. The supernatant fraction (contaille(l 15-18 mng of l)rotein per ml and all of the lertinent activitie,. Extracts lprelpared in this manner wvere diluted as re(luired into p1-1 7.5 buffer containing 1.2 MI ammonium sulfate (anid 50 nmM l)otassium phosphate.
In an effort to find out whether CoA would laffect other ''con(lensinlg" enzymes in a similar manner, we (Ieci(le(l to
Abbreviation: Nbs2, 5,5'-dithiobis(2-nitrobenzoic acid). * Author to whom requests for reprints should be addressed.
1802
Proc. Nat. Acad. Sci. USA 72
1803
CoA-Mediated Inactivation of Condensing Enzymes
(1975)
TABLE 1. Effect of coenzyme A and other compounds on a-isopropylmalate synthase, homocitrate synthase, and citrate synthase activities in crude extract Relative activity remaining after 10 min
a-IsopropylHomocitrate Citrate malate synthase synthase synthase (%) (%) (%) Additions 90 ± 8 100±zt 3 95 ±t 5 None 108±4 19±4 17±3 11 AMCoA 109 ± 7
Reversible, Coenzyme-A-Mediated Inactivation of Biosynthetic Condensing Enzymes in Yeast: A Possible Regulatory Mechanism [a-isopropylmalate syntliase (2-isopropylmalate synthase)/homocitrate synthase/citrate (si)-synthase/ channeling of acetyl-coenzyme A]
JAMES W. TRACY AND GUNTER B. KOHLHAW* Department of Biochemistry, Purdue University, West Lafayette, Indiana 47907
Communicated by Herbert C. Brown, February 21, 1975 ABSTRACT a-Isopropylmalate synthase [3-hydroxy-4methyl-3-carboxyvalerate 2-oxo-3-mnethylbutyrate-lyase (CoA-acetylating); EC 4.1.3.12], the enzyme catalyzing the first committed step in leucine biosynthesis, and homocitrate synthase [3-hydroxy-3-carbox'-adipate 2-oxoglutarate-lyase (CoA-acetylating); EC 4.1.3.21], the first enzyme in lysine biosynthesis in yeast, are rapidly inactivated in the presence of low concentrations of coenzyme A, a product of both reactions. Closely related compounds like 3'dephospho-coenzyme A or oxidized coenzyme A are almost without effect, as are other sulfhys-dryl compounds. Citrate (si)-synthase [citrate oxaloacetate-lyase (pro-3S-CH2COO- - acetyl-CoA); EC 4.1.3.7] appears to be completely resistant against inactivation by coenzyme A. Ilnactivated a-isopropylmalate and lhomocitrate synthases can be reactivated by dialysis, but not by adding excess substrate. Protection against coenzyme-A-mediated inactivation is provided by relatively high concentrations of the a-ketoacid substrate or the specific end product inhibitor of each of the two enzymes. The coenzyme-A-mediated inactivation of a-isopropylmalate synthase has been more closely investigated. It requires the presence of divalent metal ions, with Zn ++ being most effective. The inactivation does not require molecular oxygen. It occurs in the presence of low concentrations of substrates and is observed in toluene-treated cells. These results, together with evidence that a-isopropy-lmalate syntlhase and lhomocitrate synthase are located in the mitochondria, suggest a mechanism by which increasing intra-mitochondrial coenzyme A concentrations might serve as a signal of decreasing acetyl-coenzyme A levels, triggering a temporary inactivation of biosynthetic acetyl-coetizyme A-consumnig reactions in order to channel the available acet-l-coenzy-me A into the citrate cycle.
inivestigate homocitrate and citrate synthase with respect to their CoA sensitivity. Homocitrate synthase [3-hydroxy-3carl)oxyadlipate 2-oxoglutarate-lvase (CoA-acetylating); EC 4.1.3.21] catalyzes the condensation of a-ketoglutarate and acetyl-CoA, thereby initiating the biosynthesis of lysine via the a-aminoadipate pathway. The enzyme is present in yeast (2). We report here that homocitrate synithase is also specificallv inactivated by CoA. By contrast, citrate synthase [citrate oxaloacetate-ly ase (pro-3S-CH2COO- acetyl-CoA); EIC 4.1.3.7] is not affected by CoA, even at concentrations far above those effective with the other enzymes. Results from exl)eriments designed to mimic in vivo conditions suggest that the (oA inactivation could occur in the living cell. Support for a possible physiological significance of the selective effect of (oA comes fromi evidence that both a-isopropyllmalate and homnocitrate svntlhase are located in the mitochondria (see
Discuission). A brief account of some of the results reported here has been
lresenite(l (3). M~NIATERIALS AND METHODS
Chemicals. CoA (90%lpure), oxidized CoA, 3'-dephospho(oA, andl (1,N6-etheno)-CoA were purchase(I from P & L lBiochemicals, Inc. The purity of CoA as supplied was confirmed by a stoichiomnetric assay using phosphotransacety'lase from Clostridiinm kliiyveri (4) an(l by titration of free sulfhydryl groups with 5,5'-ditlhiobis(2-niitrobenizoic acid) (Nbs2). a l)rep)ared by the quantitative acetylation of Acetyl-CoA -as (CoA with acetic anhvdride after the method of Simon and Shlemlin (5). Organismi. Comimercially obtained baker's yeast (3Budweiser ye\last, Anlheuser-Buschl, Inlc., St. Louis, MIo.) was used
The enzyme a-isolprol)plmalate svnthase [3-hvdroxv-42-oxo-3-metlhvlbutv rate-lv ase (CoA-acetylating); EC 4.1.3.12] catalyzes the first reaction in the leucine biosynthetic pathway, i.e., the formationl of aisoprol)lmalate from acetvl-CoA and a-ketoisovalerate via an aldol-condensation-tvpe reac tioli. P'revious work in this laboratory on the l)rol)erties of yeast ca-isol)ro)vllmalate synthase led to the recoolnitioni of a strong inactivating effect of free CoA which occurred wlien the enzyme watis incubated with CoA in the absence of other liganals or in the l)resence of acetyl-CoA (1). a-Ketoisovalerate and the feedback inhibitor, leucine, l)artially l)rotecte(l the enzyme against CoA inactivationl, but subsequent addition of substrates (lid not reverse it. The inactivating effect was sl)ecific for CoA and was not seen with other sulfhvd(lrvl or a(lenvlate comlpoundls.
methyl-3-carboxyvalerate
throuighout these studies.
Cell Disrtiption. (Crude extracts were lprepared by suspending 5 g of cells (wet weight) in 10 ml of 50 mMA potassium phosl)hate buffer, p)lI 7.5, containing 1.5 mM\ phenylmethylsulfonll fluoride an(l passing the susl)ension through a chilled (40) Aminliico French pressure cell at 20,000 pounds/inch2 (180 il)a). To thedisrultel suspension were added 20 ml of IpII 7.5 buffer containing 2.4 MI ammonium sulfate, 50 mM\ potassium l)hol)halte, and 1.5 mi \l phenylmethyl-sulfonl fluoride. After stirring for 30 min at 00, the mixture was centrifuged for 20 min at 30,000 X g. The supernatant fraction (contaille(l 15-18 mng of l)rotein per ml and all of the lertinent activitie,. Extracts lprelpared in this manner wvere diluted as re(luired into p1-1 7.5 buffer containing 1.2 MI ammonium sulfate (anid 50 nmM l)otassium phosphate.
In an effort to find out whether CoA would laffect other ''con(lensinlg" enzymes in a similar manner, we (Ieci(le(l to
Abbreviation: Nbs2, 5,5'-dithiobis(2-nitrobenzoic acid). * Author to whom requests for reprints should be addressed.
1802
Proc. Nat. Acad. Sci. USA 72
1803
CoA-Mediated Inactivation of Condensing Enzymes
(1975)
TABLE 1. Effect of coenzyme A and other compounds on a-isopropylmalate synthase, homocitrate synthase, and citrate synthase activities in crude extract Relative activity remaining after 10 min
a-IsopropylHomocitrate Citrate malate synthase synthase synthase (%) (%) (%) Additions 90 ± 8 100±zt 3 95 ±t 5 None 108±4 19±4 17±3 11 AMCoA 109 ± 7
