Saccharomyces cerevisiae*

Transcription

1 THE JOURNAL OF BIOLOGICAL CHEMISTRY 1988 by The American Society for Biochemistry and Molecular Biology, Inc. Vol. 263, No. 29, Issue of October 15, pp , 1988 Printed in U.S.A. Purification and Characterization of an Na-Acetyltransferase from Saccharomyces cerevisiae* Fang-Jen S. Lee$#, Lee-Wen Lin$#, and John A. Smith$n(( (Received for publication, April 11, 1988) From the Departments of $Molecular Biology and llpathology, Massachusetts General Hospital, and Departments of Genetics and IIPathology, Haruard Medical School, Boston, Massachusetts 2114

2 Na-Acetyltransferase, which catalyzes the transfer of an acetyl group from acetyl coenzyme A to the a- NHz group of proteins and peptides, was isolated from Saccharomyces cerevisiae and demonstrated by protein sequence analysis to be NHz-terminally blocked. The enzyme was purified 4,6-fold to apparent homogeneity by successive purification steps using DEAE-Sepharose, hydroxylapatite, DE52 cellulose, and Affi-Gel blue. The M, of the native enzyme was estimated to be 18, f 1, by gel filtration chromatography, and the M, of each subunit was estimated to be 95, f 2, by sodium dodecyl sulfatepolyacrylamide gel electrophoresis. The enzyme has a ph optimum near 9., and its PI is 4.3 as determined by chromatofocusing on Mono-P. The enzyme catalyzed the transfer of an acetyl group to various synthetic peptides, including human adrenocorticotropic hormone (ACTH) (1-24) and its [Phe ] analogue, yeast alcohol dehydrogenase I (1-24), yeast alcohol dehydrogenase I1 (1-24), and human superoxide dismutase (1-24). These peptides contain either Ser or Ala as NH2- terminal residues which together with Met are the most commonly acetylated NHz-terminal residues (Person, B., Flinta, C., von Heijne, G., and Jornvall, H. (1985) Eur. J. Biochem. 152, ). Yeast enolase, containing a free NHz-terminal Ala residue, is known not to be Na-acetylated in vivo (Chin, C. C. Q., Brewer, J. M., and Wold, F. (1981) J. Biol. Chem. 256, ), and enolase (1-24), a synthetic peptide mimicking the protein s NHz terminus, was not acetylated in vitro by yeast acetyltransferase. The enzyme did not catalyze the Nu-acetylation of other synthetic peptides including ACTH(11-24), ACTH(7-38), ACTH(18-39), human &endorphin, yeast superoxide dismutase (1-24). Each of these peptides has an NHz-terminal residue which is rarely acetylated in proteins (Lys, Phe, Arg, Tyr, Val, respectively). Among a series of divalent cations, Cu2+ and Zn2+ were demonstrated to be the most potent inhibitors. The enzyme was inactivated by chemical modification with diethyl pyrocarbonate and N-bromosuccinimide.

3 Purification and Characterization of an N*-Acetyltransferase TABLE 1 Purification of N"-acetyltransferase from S. cerevisiae SteD Activity units Protein w Specific activityyield units/mg Purification % -fold 1.1. Crude extract ,7 3,2 2. DEAE-Sepharose 17"(.2M5.1 KCI) 8.7 3,7132,2 24"M 24.5 KCI) 41.81,47 61,5 3. DEAE-Sepharose ( ,3 4. Hydroxylapatite 5. DE52-cellulose 1, ,7 Affi-Gel blue 7, ,16 6.4,6 An apparent inhibitor was removed during thesechromatographic steps. ACTH', as well as synthetic peptides based on the sequences superoxide dismutase from human and alcohol dehydrogenase I, alcohol dehydrogenase 11, enolase, and superoxide dismutase from yeast EXPERIMENTAL PROCEDURES~ RESULTS Homogeneityand Molecular Properties-An acetyltransferase was purified to apparent homogeneity from S. cerevisiae according to the procedure described under "Experimental Procedures." As shown in Table 1,a multiple step purification

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9 I E. I - 4._ c z > 'E 2 4 ) E, 1 N c W H E 2 g 1 N 6 Fraction Number

10 I I - E 6 c &, 2 E, r 4 W 1 on 2 Fraction Number

11 T Fraction Number E E N a

12 T 4 I Fraction Number

13 I o -. E z A I L ui Fraction Number.

14 s based on the sequences mutase from human and hydrogenase 11, enolase, t. 25 CEDURES~ erties-an acetyltransfereneity from S. cerevisiae ed under "Experimental multiple step purification a 4,6-fold purification. ly.1% of total cellular lyacrylamide gel electroblue-stained band with e 6). Gel filtration chrohows that the M,of the k 1, (Fig. 7). These ansferase is composed of MonoP revealed a single ssays fordetermining the tyltransferase were pernd the yeast acetyltransat temperatures from 3 occurred after 1 min at e when stored at 4 "C in Under these conditions approximately 4-6 days. ng at all stages of purifiactivity per freeze-thaw FIG.6. Sodium dodecyl sulfate-polyacrylamidegel electrophoresis of purified yeast acetyltransferase. Theelectrophoresis was performed according to themethod of Laemmli (3)using an 8% gel. The gel was stained with Coomassie Blue. Lane I, crude extract; lane 2, DEAE-Sepharose (.2 M KCl) pool; lane 3, DEAE-Sepharose (.5to.5 M KC1) pool; lane 4, hydroxylapatite pool; lane 5, DEAEcellulose pool; lane 6, Affi-Gel blue pool; lane 7, molecular weight standards (from the top): myosin (25,),E. (116,),rabbit muscle phosphorylase (97,),bovine serum albumin (66,),and egg albumin (45,).

15 6 Acetyltransferase z h z 5- -I 4 I B Ve /Vo

16 I B 2 4

17 Purification and Characte IO I I. I. I. I ' 1 ' Temperature ( "C ) Q HEPES + K- PO^ +- CHES * CAPS PH

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19 Table 3 I n. I.1."." I2 ~.. I M)

20 2-mrraptoethanol DlT NEM L4.4 IAM pcmb TNBS Succmc anhydnds N~acetylrm>dmlc s 5 I1 I1 5 SO I.o I.o 1. I O 1. I.o loo I.o I.o too I IO Ill x x1 5s I(K1 63

21 TABLE 5 Relative activity of yeast acetyltransferase for the NO-acetylation of synthetic peptides and histones Substrate Activity' % ACTH(1-24) Ser-Tyr-Ser-Met-Glu-His-Phe-Arg-Trp- Gly-Lys-Pro-Val-Gly-Lys-Lys-Arg-Arg- Pro-Val-Lys-Val-Tyr-Pro [Phe'] ACTH(1-24) Ser-Phe-Ser-Met-Glu-His-Phe-Arg-Trp- Glxys-Pro-Val-Gly-Lys-Lys-Arg-Arg- Pro-Val-Lys-Val-Tyr-Pro ACTH(11-24) Lys-Pro-Val-Gly-Lys-Lys-Arg-Arg-Pro- Val-Lys-Val-Tyr-Pro ACTH(7-38) Phe-Arg-Trp-Gly-Lys-Pro-Val-Gly-Lys- Lys-Arg-Arg-Pro-Val-Lys-Val-Tyr-Pro- Asn-Gly-Ala-Glu-Asp-Glu-Ser-Ala-Glu- Ala-Phe-Pro-Leu-Glu ACTH(18-39) Arg-Pro-Val-Lys-Val-Tyr-Pro-Asn-Gly- Ala-Glu-Asp-Glu-Ser-Ala-Glu-Ala-Phe- 2+2 Tyr-Gly-Gly-Phe-Met-Thr-Ser-Glu-Lys- Ser-Gln-Thr-Pro-Leu-Val-Thr-Leu-Phe- Lys-Asn-Ala-Ilu-Ilu-Lys-Asn-Ala-Tyr- Lys-Lys-Gly-Glu Alcohol dehydrogenase I(1-24) (yeast) Ser-Ile-Pro-Glu-Thr-Gln-Lys-Gly-Val-Ile- Phe-Tyr-Glu-Ser-His-Gly-Lys-Leu-Glu- Tyr-Lys-Asp-Ile-Pro hi- Alcohol dehydrogenase II(1-24) (yeast) Ser-Ile-Pro-Glu-Thr-Gln-Lys-Ala-Ile-Ile- Phe-Tyr-Glu-Ser-&-Gly-Lys-Leu-Glu- - His-Lys-Asp-Ile-Pro Superoxide dismutase( 1-24) (yeast) Val-Gln-Ala-Val-Ala-Val-Leu-Lys-Gly- Asp-Ala-Gly-Val-Ser-Gly-Val-Val-Lys- Phe-Glu-Gln-Ala-Ser-Glu Superoxide dismutase( 1-24) (human) 86 * 6 Ala-Thr-Lys-Ala-Val-Cys-Val-Leu-Lys- Gly-Asp-Gly-Pro-Val-Gln-Gly-Ser-Ile- Asn-Phe-Glu-Gln-Lys-Glu Enolase(1-24) (yeast) 4+2 Ala-Val-Ser-Lys-Val-Tyr-Ala-Arg-Ser-Val- Tyr-Asp-Ser-Arg-Gly-Asn-Pro-Thr-Val- Glu-Val-Glu-Leu-Thr Histone (lysine-rich)(calf thymus) Histone (arginine-rich)(calf thymus) "Data reported as mean activity + S.D. (n = 3-5).