June 1973 issue of Proc. Nat. Acad. Sci. USA 70, , Fig. 5 (p. 1792) contains an error. The published sequence

Transcription

1 Proc. Nat. Acad. Sci. USA 70 (1973) Correction. In the article "Aminoacid Sequence of Dogfish M4 Lactate Dehydrogenase," by aylor, S. S., Oxley, S. S., Allison, W. S. & Kaplan, N. O., which appeared in the Correction 2467 June 1973 issue of Proc. Nat. Acad. Sci. USA 70, , Fig. 5 (p. 1792) contains an error. he published sequence in the pig H4 peptide is as indicated in the figure below VAL-ILE-GLY-GLN-HIS-GLY-ASP-SER-VAL-PRO-SER-VAL-RP- ILE-LEU-GLY-GLU-HIS-GLY-ASP-SER-VAL-PRO-SER-VAL-RP FIG. 5.

2 Proc. Nat. Acad. Sci. USA Vol. 70, No. 6, pp , June 1973 Aminoacid Sequence of Dogfish M4 Lactate Dehydrogenase (glyceraldehyde-3-phosphate dehydrogenase/lactic acid yeast and liver alcohol dehydrogenases) SUSAN S. AYLOR, SUSANNA S. OXLEY, WILLIAM S. ALLISON, AND NAHAN 0. KAPLAN he Department of Chemistry, University of California, San Diego, La Jolla, Calif Contributed by Nathan 0. Kaplan, March 19, 1973 ABSRAC About 80% of the aminoacid sequence of dogfish (Squalus acanthius) M4 lactate dehydrogenase (EC ) has been elucidated. Several sequence homologies with peptides from pig H4 and pig M4 lactate dehydrogenase are identified. Histidine 195 is homologous to the essential histidine residue in pig H4 lactate dehydrogenase. Similarities in the sequence around the "essential" cysteine residue of lactate dehydrogenase, glyceraldehyde-3- phosphate dehydrogenase, and yeast and liver alcohol dehydrogenases are delineated. Lactate dehydrogenase is a tetrameric, NAD-requiring enzyme. he subunits are identical and have a molecular weight of 36,000 (1); no cooperative effects of the subunits have been demonstrated (2, 3). At least two types of lactate dehydrogenase are found in significant amounts in most species; the M4 isozyme predominates in skeletal muscle and the H4 isozyme in more aerobic tissues such as heart and kidney cortex (4, 5). he two types are distinguishable from one another by electrophoresis, aminoacid composition, immunological methods, and kinetic properties (5-7). hey show significant differences in their response to substrate inhibition, and this difference may be the basis of distinct physiological roles (5, 8). Many lactate dehydrogenases have been characterized to varying extents, and it appears that homologous isozymes of the same enzyme type from various species are more similar in their properties to each other than are the two corresponding forms in any one given species. Nevertheless, mixed hybrids of M and H subunits are observed in vivo (4, 5) and can be readily formed in vitro (9) suggesting a considerable degree of structural similarity between the two proteins. he sequence reported here has been elucidated for the M4 isozyme from dogfish (Squalus acanthius) muscle. his same dogfish M4 isozyme has been characterized by x-ray diffraction (10-13), and the correlation of the aminoacid sequence with the crystallographic structure will be discussed in more detail (33). he enzymatic activity of lactate dehydrogenase is inhibited by most sulfhydryl reagents, and there are four functionally significant cysteine residues per tetramer (14). A dodecapeptide containing this cysteine residue has been sequenced from several lactate dehydrogenases (1, 15, 16). he sequences of the N-terminal 18 residues of dogfish M4 lactate dehydrogenase (1), of several small peptides from pig H4 lactate dehydrogenase (17), and of a peptide containing an essential histidine residue have been reported (11, 18). Preliminary evidence for involvement of one, and possibly as many as three, arginine residues (19) and of one tyrosine residue (20, 21) per subunit in enzymatic activity has been presented. MAERIALS AND MEHODS Purification and Peptide Isolation. Dogfish M4 lactate 1790 dehydrogenase was purified by the procedure of Pesce et al. (6). An aminoacid analysis of this protein is given in able 1. Before proteolytic digestion, the homogeneous protein was carboxymethylated in 10 mm ris HCl (ph 8.3)-0.5 mm EDA-8 M urea with a 2-fold excess of [14C]iodoacetic acid for 4 hr at room temperature (240), followed by extensive dialysis. he sequence was determined primarily from peptides isolated after tryptic and chymotryptic digestion. hirty-five tryptic peptides were purified and sequenced out of an expected 40 pflptides; the remaining peptides were partially sequenced. he carboxymethylated lactate dehydrogenase was also treated with maleic anhydride, which specifically blocks the lysine residues (22). ryptic digestion of this maleated protein yields 10 peptides, seven of which were completely sequenced. Finally, the peptide fragments resulting from treatment with cyanogen bromide (23) were characterized. Since there are 11 methionine residues per subunit, 12 peptides should be obtained after cyanogen bromide treatment; the sequence of 10 of these peptides was elucidated. All sequencing was done by the dansyl-edman procedure (24). Dansyl-amino acids were identified by thin-layer chromatography on 4 X 4 inch polyamide sheets (25). Aminoacid compositions were determined on a Beckman model 120C amino acid analyzer. Peptides were purified by numerous methods, including both Sephadex and ion exchange column chromatography, paper electrophoresis at ph 6.5, 1.9, 3.5, and 8.9, and paper chromatography in butanolacetic acid-water-pyridine 15:3:12: 10. Amide identification was based on electrophoretic mobilities at ph 6.5 (26). he Numbering of the Polypeptide Chain is that proposed by Rossmann et al. (11) based on the 2.5-A map of the apoenzyme. Subsequent improvement of the crystallographic resolution (13) as well as sequence information has shown that residues 21, 82, and 104 in the amino terminal region should be deleted and an extra residue should be inserted at position 245. he total number of residues is therefore somewhat less than the 331 originally proposed. Nevertheless, until the entire sequence is complete, this numbering scheme shall be adhered to. Of the tentative total of 329 aminoacid residues per subunit, 263 are reported here in three portions: the amino terminal region (1-115), residues , and the carboxy terminal region ( ). RESULS AND DISCUSSION Amino erminal Region (1-115). he sequence of this region was established from proteolytic digests (Fig. 1). he 13 tryptic peptides in this region have been sequenced completely except for the region in parentheses [residues 29-33],

3 Proc. Nat. Acad. Sci. USA 70 (1973) Lactate Dehydrogenase Sequence 1791 ACEYL- 20 HR- ALA- LEU- LYS- ASP- LYS- LEU- ILE- GLY- HIS- LEU- ALA- HR- SER- GLN- GLU- PRO- ARG- SER- YR- ASN- LYS- I LE- HR- VAL- VAL- GLY(Cys, M-1-4 C Ala,Asx,Val,GIy )ME- ALA- ASP- ALA- ILE- SE R- VAL- LEU- ME- LYS- ASP- LEU- ALA- ASP- GLU- VAL- ALA- LEU- VAL- ASP- VAL- ME- GLU- ASP- LYS- -5-6,7 CNBr-2 CNBr LEU- LYS- GLY- GLU- ME- ME- ASP- LEU- GLU- HIS- GLY- SER- LEU- PHE- LEU- HIS- HR- ALA- LYS- ILE- VAL- SER- GLY- LYS- ASP-YR- SER- VAL- I8i-8 Ii -9,10 CNBr-4 I M- CNBr SER- ALA- GLY- SER- LYS- LEU- VAL- VAL- ILE- H R- ALA- GLY- ALA- ARG- GLN- GLN- GLU- GLY- GLU- SER- ARG- LEU- ASN- LEU- VAL- GLN- ARG FIG. 1. Aminoacid sequence of the N-terminal region of dogfish M4 lactate dehydrogenase. he numbering of the residues is as indicated in the text; the deletions are designated as blank spaces at positions 21, 82, and 104. ype of digest from which the peptide was isolated; = tryptic; C = chymotryptic; CNBr = cyanogen bromide; M = tryptic peptide from maleated protein. Positions 16 and 17 have been modified from the original reported sequence (1). C-11 where only the composition has been established. Four CNBr fragments from this region were also characterized, and with these peptides, eight of the 12 overlapping regions of the tryptic peptides were determined. Identification of the acetyl substitution of the amino terminal threonine was confirmed by nuclear magnetic resonance spectroscopy of the peptide acetyl-hr-ala-leu*. his first third of the molecule is involved primarily in coenzyme binding. In addition, the section extending from residue forms the loop that differs significantly in Conformation in the crystal structures of the apoenzyme and ternary complex (11). It is interesting to note that two of the tryptic peptides in this portion of the polypeptide chain, -12 and -13, are homologous to peptides isolated from pig H4 and M4 lactate dehydrogenase (17) (Fig. 2). Aminoacid Residues his region (Fig. 3) appears to contain many of the residues important 'for lactate and pyruvate binding. An unusual feature of the total sequence is the uneven distribution of arginine residues. Each subunit contains only nine arginine residues, and this fragment contains four of these arginines in a cluster of consecutive arginine tryptic peptides. he order of these fragments was established by a single cyanogen bromide peptide. he "essential" cysteine residue is found in this region and from x-ray crystallographic data was identified as residue 165 (11). he tryptic peptide containing this cysteine has been isolated from several different lactate dehydrogenases, including both M4 and H4 isozymes, and in all cases the sequence has been closely conserved (15, 16). If a single deletion is assumed to have occurred in the lactate dehydrogenase sequence, a reasonable comparison can be made of the sequence of this peptide with the sequence sumrounding the "essential" cysteine in yeast alcohol dehydrogenase (27), horse-liver alcohol dehydrogenase (27, 28), and glyceraldehyde-3-phosphate dehydrogenase (29, 30) (Fig. 4). If, in addition, consideration is given to those amino acids whose codons may be related by a single nucleotide base change in the mrna sequence (16), the similarities in the sequences of these peptides become even more apparent. he correlation of functionally similar amino acids is also reasonably good. In lactate dehydrogenase the "essential" cysteine is not one of the most reactive cysteines in the molecule (16), and, sur- * Morelli, R. & Allison, W. S., unpublished results. prisingly, correlation of the sequence with the crystal structure indicated that this cysteine does not participate directly in either substrate or coenzyme binding (33). In contrast, the essential cysteine of glyceraldehyde-3-phosphate dehydrogenase is extremely reactive and forms an S-acyl-enzyme intermediate (31). Woenckhaus et al. inactivated pig H4 lactate dehydrogenase with 3-(2-bromo-1-[14C]acetyl) pyridine (18). From this inactivated enzyme they isolated a single peptide containing a modified radioactive histidine derivative. Adams et al., on the basis of the sequence of this peptide, subsequently identified this histidine as residue 195 (11). In Fig. 5, the Woenckhaus peptide is compared with the corresponding sequence from dogfish M4 lactate dehydrogenase. his, too, is a region of the molecule where the sequence in these two proteins is highly conserved. Carboxy-erminal Sequence ( ). Nine tryptic peptides were isolated and sequenced from the C-terminal region of the protein (Fig. 6). Chymotryptic peptides established the overlapping portion of six of the peptides. hree cyanogen bromide fragments accounted for the final 69 residues of this region, and with these cyanogen bromide peptides the order of the remaining tryptic peptides was determined. his sequence is compared with the known C-terminal sequence of pig H4 lactate dehydrogenase (32) (Fig. 7). Eleven of the final 14 residues are identical. An insertion in ABLE 1. Aminoacid composition in mol per subunit (36,000 molecular weight) of carboxymethylated dogfish M4 lactate dehydrogenase LYSINE 29.6 ASPARIC ACID 33.7 HISIDINE 10.7 HREONINE 13.0 ARGININE 8.8 SERINE 26.2 GLUAMIC ACID 26.0 PROLINE 12.0 GLYCINE 24.9 ALANINE 19.7 VALINE 33.1 MEHIONINE 10.5 CM-CYSEINE 5.7 ISOLEUCINE 19.0 LEUCINE 33.1 YROSINE 7.1 PHENYLALANINE 6.6 his composition is an average of multiple hydrolysates. Duplicate samples each containing 200,ug of protein were hydrolyzed under reduced pressure at 105 in 6 N HOC, for 24, 48, and 72 hr.

4 1792 Biochemistry: aylor et al. Proc. Nat. Acad. Sci. USA 70 (1978) 110 -ARG -GLN -GLN -GLU -GLY -GLU -SER -ARG -LEU -ASN -LEU -VAL -GLN -ARG - -ARG GLN -GLN -GLU -GLY -GLX -SER -ARG LEU -ASN -LEU -VAL -GLN -ARG i I - I -ARG GLN -GLN -GLU -GLY -GLX -SER -ARG LEU -ASN -LEU -VAL -GLN -ARG PIG M4 i ---I - i FIG. 2. Possible sequence homologies in the peptide fragment beginning at residue 101. he positioning of the pig heart and pig muscle peptides (17) was established here by alignment with the dogfish M4 sequence LYS- GLU- LEU- HIS- PRO- GLU- LEU- GLY-HR-ASP- LYS- ASN- LYS- GLN- ASP-RP- LYS- LEU- IF- CNBr SER-GLY- LEU-PRO-ME-HIS-ARG- ILE- ILE-GLY- SER- GLY--CYS-ASN- LEU- ASP-SER-ALA- - *1 CNBr 180 ARG-PHE-ARG-YR-LEU-ME-GLY-GLU-ARG-LEU-GLY-VAL-HIS-SER (Cys, Leu, Val, lie. I I I Gly)RP-VAL- ILE-GLY-GLN-HIS-GLY-ASP-SER-VAL-PRO-SER-VAL-RP-ME(Asx. CNBr C FIG. 3. Aminoacid sequence of residues in dogfish Mt lactate dehydrogenase. IE3 lie GLYSERGLY- CYS ASP AA AG -PHE RG-- LACAE DEHYDROGENASE rvlkilile ILVSERG Y - CYS AS {E 4J {E ALA A LACAE DEHYDROGENASE '17) i IE -r ]-[S:]-R1{ [[ I-SER -CYS -HR AS ] -LEU -ALAU-PRO LEGI GLYCERALDEHYDE-3-P DEHYDROGENASE* LLYSJ -LM~iE,rALI AH LEE -ILE -CYS -ARG -E ASP -ASP -HIS -VAL -HR 4SER- HORSE LIVER ALCOHOL DEHYDROGENASE (28.29) --YR { --VAL -YS n'-hr A --HIS -ALA-RP HS YEAS ALCOHOL DEHYDROGENASE (28) FIG. 4. Comparison of the sequence around the "essential" cysteine (underlined) of several dehydrogenases. A single deletion is shown in the lactate dehydrogenase sequence between Gly-164 and Cys-165, which tends to maximize any possible homologies. indicates those residues that are, identical; indicates those aminoacid residues whose codon could differ from the corresponding dogfish M4 lactate dehydrogenase by a single nucleotide base change in the mrna sequence. *Identical sequence for yeast (29), pig muscle (29), and rabbit muscle (30) VAL -ILE -GLY -GLN -HIS -GLY -ASP -SER -VAL -PRO -SER -VAL -RP- VAL -ILE -GLY -GLU -HIS -GLY -ASP -SER -VAL -PRO -SER -VAL -RP FIG. 5. Sequence homologies around histidine 195. *Essential histidine residue in pig 1 lactate dehydrogenase (10, 18) SE R- VAL- ALA- ASP- LEU- ALA- GLN- H R- LE- ME- LYS- ASN- LEU- CYS- ARG- VAL- HIS- PRO- VA.- SER- - - I - C I:C I CNBr HR- ME- VAL- LYS- ASP- PHE- YR- GLY- ILE- LYS- ASP- ASN- VAL- PHE- LEU-SER- LEU-PRO-CYS- VAL-,- -, - - cc - C LEU- ASN- ASX- GLY- LE- SE R- HIS-C Ys - ASN- LE- VAL- LYS- ME- LYS- LEU- LYS- PRO- ASP- GLU- GLU- II - c,i GLN GLN- LEU-GLN- LYS-SER-ALA-HR-HR- LEU-RP-ASP-ILE-GLN-LYS-ASP-LEU-LYS-PHE II Hi c C c I FIG. 6. Aminoacid sequence of the carboxy-terminal region of dogfish M4 lactate dehydrogenase.

5 Proc. Nat. Acad. Sci. USA 70 (1978) Lactate Dehydrogenase Sequence SER -HIS -Cys -ASN -ILE -VAL -LYS -ME -LYS LEU LYS -PRO - ARG -LEU -LYS -ASP -ASP -GLU -VAL -ALA -GLN LE L3 -GLY - PIG I ASP -GLU -GLU -GLN -GLN -LEU FG-LN Ls-ER AA-HR - LEU -HR -SER -ASN -VAL -ILE GLN YS-ASN SER AL-ASP H ASP -{3 GL Y LYSE -PHE HR EU -GLY -fj EGNL VS-gLEU -ASP -LEU FIG. 7. Comparison of the C-terminal sequence of lactate dehydrogenase from pig H4 (31) and dogfish M4 isozymes. indicates those residues that are identical. the pig H4 sequence after residue 317 maximizes the homology; nevertheless, it is clear that the sequence becomes more divergent in the region extending from residue CONCLUSION Although the sequence in two regions of the molecule remains to be confirmed, it is already possible to align the known sequence of dogfish M4 lactate dehydrogenase with the crystallographic structure. From the crystallographic data and various chemical and kinetic information it has also been possible to identify precisely many of the specific residues important for substrate and coenzyme binding. A preliminary comparison of sequence homologies between dogfish M4 and pig H4 and M4 lactate dehydrogenase suggests that the similarities may be extensive even though they are different types isolated from different species. his research was supported by Research Grants GM 16979, GM 13901, and CA from the National Institutes of Health. S.. was also the recipient of a Public Health Service Postdoctoral Fellowship CA and a Career Development Award GM from the National Institutes of Health. 1. Allison, W. S., Admiraal, J. & Kaplan, N. 0. (1969) J. Biol. Chem. 244, Heck, H. d'a. (1969) J. Biol. Chem. 244, Schwert, G. W., Miller, B. R. & Peanasky, R. J. (1967) J. Biol. Chem. 242, Appella, E. & Markert, C. L. (1961) Biochem. Biophys. Res. Commun. 6, Cahn, R. D., Kaplan, N. O., Levine, L. & Zwilling, E. (1962) Science 136, Pesce, A. J., McKay, R. H., Stolzenbach, F. G., Chan, R. D. & Kaplan, N. 0. (1964) J. Biol. Chem. 239, Pesce, A. J., Fondy,. P., Stolzenbach, F. G., Castillo, F. & Kaplan, N. 0. (1967) J. Biol. Chem. 242, Everse, J., Berger, R. L. & Kaplan, N. 0. (1970) Science 168, Chilson, 0. P., Costello, L. A. & Kaplan, N. 0. (1965) Biochemistry 4, Adams, M. J., Ford, G. C., Leikoek, R., Lentz, P., Jr., McPherson, A., Jr., Rossmann, M. G., Smiley, I. E., Schevitz, R. W. & Wonacott, A. J. (1970) Nature 227, Rossmann, M. G., Adams, M. J., Buehner, M., Ford, G. C., Hackert, M. L., Lentz, P. J., Jr., McPherson, A., Jr., Schevitz, R. W. & Smiley, I. E. (1971) Cold Spring Harbor Symp. Quant. Biol. 36, Adams, M. J., Buehner, M., Chandrasekhar, K., Ford, G. C., Hackert, M. L., Liljas, A., Lentz, P., Jr., Rao, S.., Rossmann, M. G., Smiley, I. E. & White, J. L. (1972) in Protein- Protein Interactions, eds. Jaenicke, R. & Helmreich, E. (Springer-Verlag, New York), p Adams, M. J., Liljas, A., Rossmann, M. G. & McPherson, A., Jr. (1973) J. Mol. Biol., in press. 14. DiSabato, G. & Kaplan, N. 0. (1963) Biochemistry 2, Fondy,. P., Everse, J., Driscoll, G. A., Castillo, F., Stolzenbach, F. G. & Kaplan, N. 0. (1965) J. Biol. Chem. 240, Holbrook, J. J., Pfleiderer, G., Mella, K., Volz, M., Leskowac, W. & Jeckel, D. (1967) Eur. J. Biochem. 1, Pfleiderer, G., Woenckhaus, C. J., Jeckel, D. & Mella, K. (1970) in Pyridine Nucleotide-Dependent Dehydrogenases, ed. Sund, H. (Springer-Verlag, New York, pp ). 18. Woenckhaus, C., Berghauser, J. & Pfleiderer, G. (1969) Hoppe-Seyler's Z. Physiol. Chem. 350, Schwert, G. W. & Yang, P. C. (1972) Biochemistry 11, DiSabato, G. (1965) Biochemistry 4, Jeckel, D., Anders, R. & Pfleiderer, G. (1971) Hoppe- Seyler's Z. Physiol. Chem. 352, Butler, P. J. G., Harris, J. I., Hartley, B. S. & Leberman, R. (1959) Biochem. J. 112, Gross, E. & Witkop, B. (1962) J. Biol. Chem. 237, Gray, W. R. & Hartley, B. S. (i963) Biochem. J. 89, and 59P. 25. Hartley, B. S. (1971) Biochem..J. 119, Offord, R. (1966) Nature 211, Harris, I. (1964) Nature 203, Li,. K. & Vallee, B. L. (1964) Biochemistry 3, Perham, R. N. & Harris, J. I. (1963) J. Mol. Biol. 7, Harris, J. I., Meriwether, B. P. & Park, J. N. (1963) Nature 198, Krimsky, I. & Racker, E. (1955) Science 122, Mella, K., orff, J. J., Folsohe, E. h.j. & Pfleiderer, G. (1969) Hoppe-Seyler's Z. Physiol. Chem. 350, Adams, M. J., Buehner, M., Chandrasekhar, K., Ford, G. C., Hackert, M. L., Liljas, A., Rossmann, M. G., Smiley, I. E., Allison, W. S., Everse, J., Kaplan, N. 0. & aylor, S. S. (1973) Proc. Nat. Acad. Sci. USA 70, in press. APPENDIX Electron Density Map as an Aid in the Sequencing of Peptides MARGARE J. ADAMS*, GEOFFREY C. FORD, PAUL J. LENZ, JR.t, ANDERS LILJAS, AND MICHAEL G. ROSSMANN Department of Biological Sciences, Purdue University, West Lafayette, Indiana An electron density distribution, at a nominal resolution of 2.0 X, was used as a guide and check on the positioning of selected * Present address: Department of Molecular Biophysics, Zoology Department, South Parks Road, Oxford, England. t Present address: Wallenberg laboratoriet, Uppsala Universitet, Dag Hammarskjblds vag 21, Uppsala, Sweden.