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. 14948-14955, 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
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, 523-527). 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, 1377-1384), 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.
Purification and Characterization of an N*-Acetyltransferase 14949 TABLE 1 Purification of N"-acetyltransferase from S. cerevisiae SteD 21 87 27 Activity units Protein w Specific activityyield units/mg Purification % -fold 1.1. Crude extract 1.7 17,7 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 (.5-.5 36 53.6 19,3 4. Hydroxylapatite 5. DE52-cellulose 1,5 8.58 12,7 Affi-Gel blue 7,8 1.5 8,16 6.4,6 An apparent inhibitor was removed during thesechromatographic steps. ACTH', as well as synthetic peptides based on the sequences of @-endorphin,and superoxide dismutase from human and alcohol dehydrogenase I, alcohol dehydrogenase 11, enolase, and superoxide dismutase from yeast. 1 64 25 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 116 97 66
cell Exuacnon
I E. I - 4._ c 2 3 - z > 'E 2 4 ) E, 1 N c W 2 4 6 8 121 5 4 3 H E 2 g 1 N 6 Fraction Number
I I - E 6 c - 2. 4-2 &, 2 E, 4 3 2 1 r 4 W 1 on 2 Fraction Number
T 4 1-15 1 2 Fraction Number E E N a.5.4.3.2.1.
T 4 I 3 2 1 2 1 Fraction Number
I - 3 22.2 1.o -. E z 2.8 - A I L 9 1.12.2 ui 1 5 1 15 Fraction Number.
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 116 97 66 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. coli @-galactosidase (116,),rabbit muscle phosphorylase (97,),bovine serum albumin (66,),and egg albumin (45,).
6 Acetyltransferase z h z 5- -I 4 I. 1.8 2. 2.2 2.4 2.6 2 B Ve /Vo
8 7 6 6 4 5 I B 2 4
Purification and Characte IO 8 6 4 2 I I. I. I. I ' 1 ' 1 2 3 46 5 Temperature ( "C ) 2 15 1 Q HEPES + K- PO^ +- CHES * CAPS 5 4 6 8 1 12 PH
Table 3 I n. I.1."." I2 ~.. I 2 43 84 M) 86 42 9 53 78 58 4
2-mrraptoethanol DlT NEM L4.4 IAM pcmb TNBS Succmc anhydnds N~acetylrm>dmlc s 5 I1 I1 5 SO I.o 1. 1. 1. 1. 1. I.o 1. I O 1. I.o loo I.o 1. 1 1. I.o too I IO Ill1 92 13 11x1 73 98 63 11x1 5s 94 76 41 71 I(K1 63
TABLE 5 Relative activity of yeast acetyltransferase for the NO-acetylation of synthetic peptides and histones Substrate Activity' % ACTH(1-24) 1 + 5 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) 9 + 9 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- Pro-Leu-Glu @-Endorphin(human) 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) 11 + 5 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) 12 + 4 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).