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Enzymatic trimethylation of residue-72 lysine in cytochrome c. Effect on the total structure
144
Biochimica et Biophysica Acta, 6 2 2 ( 1 9 8 0 ) 1 4 4 - - 1 5 0 © E l s e v i e r / N o r t h - H o l l a n d B i o m e d i c a l Press
BBA 38382
ENZYMATIC TRIMETHYLATION OF RESIDUE-72 LYSINE IN CYTOCHROME c EFFECT ON THE TOTAL STRUCTURE
C H A N G - S E KIM *, F. K U E P P E R S **, P. D I M A R I A , J. F A R O O Q U I , S. KIM a n d W.K. P A I K
Fels Research Institute and Department of Biochemistry, Temple University School of Medicine, Philadelphia, PA 19140 (U.S.A.) (Received June 27th, 1979)
Key words: Cytochrome c methylation; Isoelectric focusing; Space-filling model; Protein methyluse; (N. crassa, S. cerevisiae)
Summary A highly purified protein methylase III from Neurospora crassa or Saccharom y c es cerevisiae specifically methylates a single lysine residue of position 72 of horse heart cytochrome c. The enzymatically methylated cytochrome c has been separated from the unmethylated counterpart species by isoelectric focusing. Simultaneously, the pI values of these two species were found to be 9.49 and 10.03, respectively. Since methyl substitution increases the basicity associated with the e-amino group of lysine residues, the observed decrease in pI value is in opposition to the predicted increase. Space-filling models revealed the possibility of a hydrogen bond between the oxygen of amide of residue-70 asparagine and the e-amino nitrogen of residue-72 lysine in unmethylated horse heart cytochrome c. The enzymatic methylation of residue-72 lysine tends to dissociate this hydrogen bond, thereby possibly inducing the shift of 'effective charge' of the protein molecule. This paper also deals with the pI values of cytochromes c from 13 different sources, determined by the isoelectric focusing technique.
* Present address: D e p a r t m e n t of Biochemistry, Chosun University School of Medicine, Kwangju, Korea. ** Present address: Department o f Medicine, Temple University School of Medicine, Philadelphia, PA 19140, U.S.A.
145 Introduction
Protein methylation is one of the postt~anslational modification reactions, which occurs ubiquitously in many proteins involving various amino acid side chains [1,2]. In particular, DeLange et al. [3] earlier observed that cytochrome c of lower organisms have the residue-72 lysine methylated in vivo. In accordance with this observation, we have recently identified an enzyme from Neurospora crassa and Saccharomyces cerevisiae which methylates only a single lysine of position 72 of in vivo unmethylated horse cytochrome c [4--6]. The enzymatic methylation of cytochrome c facilitates the binding of this protein to mitochondria [ 7,8]. In an attempt to further understand the mechanism of the facilitation of cytochrome c binding to mitochondria by lysine methylation, the determination of the pI values of enzymatically methylated and unmethylated cytochrome c by isoelectric focusing was performed: the pI values of these two species were found to be 9.49 and 10.03, respectively. Attempts were also being made to build a space-filling model of residues 70--80 which is invariant among the various cytochromes c throughout evolution. Materials and Methods Materials. Cytochromes c of various species: horse heart, S. cerevisiae, bovine heart, pigeon heart, rabbit heart, tuna heart, Candida krusei, chicken heart, porcine heart and canine heart were obtained from Sigma Chemical Co. Cytochromes c of guanaco and mouse heart were prepared according to the method of Margoliash and Walasek [9], and N. crassa by the method of Scott and Mitchell [ 10]. S-Adenyl-L-[Me-14C]methionine (specific activity, 54.7 Ci/mol) and Ampholine (pH 9--11) were obtained from New England Nuclear and LKB, Stockholm, respectively. All other reagents were of the highest grade obtainable. Preparation o f [Me-~4C]cytochrome c. Enzymatically methylated horse heart cytochrome c was prepared according to the published method [5]. The reaction mixture contained 0.1 M glycine/NaOH buffer (pH 9.0), 5.7 mM mercaptoethanol, 0.4 mM EDTA, 30 mg horse heart cytochrome c, 40 pM S-adenosylL-[Me-~4C]methionine (100 cpm/pmol) and 20 pg of highly purified protein methylase III of N. crassa (S-adenosylmethionine:protein(lysine)N-methyltransferase, EC 2.1.1.43) in a final total volume of 1.2 ml. After 1 h incubation at 37°C, an additional 0.2 ml (20 pg) of enzyme was added, and the reaction was allowed to proceed for another 1 h. In order to remove unreacted S-adenosyl-L-[Me-laC]methionine, the reaction mixture was dialyzed against deionized water for 6 h with occasional changes. The sample was lyophilized until use.
Isoelectric focusing. Isoelectric focusing of cytochrome c from various species was performed according to the method of Vesterberg [11], usinga 110 ml column. A linear gradient was prepared by mixing light and dense solution. The light solution was made up of 59.5 ml of water and 0.5 ml of LKB Ampholine (pH 9--11, 20%). The dense solution was comprised of 28 g of sorbitol dissolved in 38.5 ml of water and 1.5 ml of Ampholine (pH 9--11, 20%). The gra-
146 dients were laid on t o p of the dense anode solution which was prepared by dissolving 12 g of sorbitol in 14 ml of water containing 0.1 ml of concentrated sulfuric acid. The cathode solution containing 0.05 g of NaOH was carefully layered on top of the sorbitol gradients. Isoelectric focusing was carried o u t in a water-jacket LKB 8100 column at 0--2°C and 800 V for 20--24 h. The column was then eluted from the b o t t o m using a Buchler multistaltic pump with a flow rate of 0.4 ml/min. Fractions of 2 ml were collected and the pH and Ass0 of each fractions were measured at room temperature. Radioactivity in each fraction was measured in an aliquot of 0.1 ml using scintillation Formula 963 (New England Nuclear Corp.). Amino acid analysis. Proteins were hydrolyzed in 6 N HC1 for 48 h at l l 0 ° C in vacuo. Amino acid analysis was carried o u t as described by Paik and Kim [12]. Results
Separation of enzymatically methylated [Me-14C]cytochrome c of horse heart from unmethylated species by isoelectric focusing Fig. I illustrates the separation of enzymatically methylated horse heart c y t o c h r o m e c from unmethylated species by isoelectric focusing. It had previously been demonstrated in this laboratory that c y t o c h r o m e c-specific protein methylase III of N. crassa methylated a single lysine residue at position 72 among 19 lysine residues present in c y t o c h r o m e c molecule and that the
10.03
°"
10.~ 10.(
400
~H ~.~" 9.¢ ~.~ ] cpm
cpm
o.(
~A~o
i o., Asso
0.2
,~ ~'~ ~
~'o ~
s6 ~
Fraction N o . focusing of enzyrnaticaliy methylated [Me-14C]cytoch~ome c o f h o r s e hesxt. Horse c was methylated i n v i t r o b y a h i g h l y p u r i f i e d N. crassa p r o t e i n m e t h y l a s e I I I w i t h S-adenosyl-L-[Me-14C]methionine. A f t e r d i a l y s i s , t]~e s a m p l e w a s a n a l y z e d o n a n e l e c t r o f o c u s i n g w i t h A m p h o H n e of pH 9--11. Methylated cytochrome c is indicated by radioactivity, and tmmethy]ated (cont r o l ) c y t o c h r o m e c b y A 5 50 • F i g . 1. I s o e l e c t r i c heart cytoch~ome
147
enzymatic methylation in vitro proceeded to a maximum of 0.5% of the total cytochrome c population [4,5]. Thus, when separated by isoelectric focusing, the methylated cytochrome c can only be visualized by its radioactivity (Fig. 1), and the unmethylated cytochrome c by the absorbance at 550 nm. As shown in Fig. 1, the major bands of radioactivity and A55onm are clearly separated by isoelectric focusing, using Ampholine pH 9--11. The isoelectric points (pI) of both methylated and unmethylated horse heart cytochromes c were determined to be 9.49 and 10.03, respectively. In order to confirm that radioactive peak is indeed methylated cytochrome c, the peak radioactivity fractions (fractions 33--35) were pooled, dialyzed and lyophilized. Amino acid analysis of the sample indicated that the radioactivity represented mostly trimethyllysine. The presence of trimethyllysine further ruled out the possibility of this peak as deamidated cytochrome c observed by Flatmark and Vesterberg in the case of beef heart cytochrome c [13]. The separation of methylated cytochrome c from unmethylated was also done using isoelectric focusing on polyacrylamide gels as described by O'Farrell et al. [14]. The pattern of separation was the same as that obtained with the preparative method (not shown). p I values o f cytochromes c from various sources Table I lists pI values of cytochrome c isolated from various sources determined by us as well as corresponding values reported in the literature. In comparing our values with the corresponding reported values, it is seen that with the exception of C. krusei and N. crassa cytochromes c our values are slightly lower. This can be attributed to the fact that our values were determined at TABLE I ISOELECTRIC POINTS (pl) OF CYTOCHROMES
c ISOLATED FROM VARIOUS SOURCES
T h e n u m b e r s in b r a c k e t s i n d i c a t e r e f e r e n c e n u m b e r s . F o r p r e s e n t w o r k p I values w e r e d e t e r m i n e d at 2 4 -+ I ° C . F o r o t h e r s ' w o r k p I values w e r e d e t e r m i n e d at 4 ° C . Sources of cytochrome c
Isoelectric point (pl) Present work
Others' w o r k
Horse h e a r t
10.03
10.65 10.9 10.35 10.7
[18] [19] [20] [21]
Mouse heart Rabbit heart Bovine heart Guanaco heart Tuna heart Canine h e a r t Porcine heart Chicken heart P i g e o n heart C. k r u s e i $. cerevisiae N. crassa Horse heart; enzymatically methylated
10.03 10.02 10.01 10.01 10.01 10.01 10.00 10.00 9.98 9.98 9.91 9.40 9.49
10.8 10.8
[19] [19]
9.8 [20] 10.4--10.5 [22] 9.34 [23]
* I t w a s f o u n d ~hat, w i t h all p r e p a r a t i o n s , t h e r e w e r e m o r e t h a n o n e b a n d i n d i c a t i n g c o n t a m i n a t i o n . T h u s , p I values o f m a j o r b a n d have b e e n t a k e n .
148 25°C as opposed to 4°C for the values determined by others (it is known that pI measurement is dependent on temperature [15]}. Looking at the cytochromes c of higher organisms, it can be seen th~t their pI values are close to 10 or little higher. The absence of in vivo methylated lysine residues have been reported in these cytochrome c species [3]. Only N. crassa and in vitro enzymatically methylated horse heart cytochromes c have pI values significantly below those of the higher organisms. Space-filling model It has previously been shown that e-N-trimethyllys.ine is the predominant product of in vitro enzymatically methylated cytochrome c along with the presence of small amounts of e-N-mono- and e-N-dimethyllysine [4,5]. Since the introduction of three methyl groups to the e-amino group of lysine residues makes the nitrogen a quaternary ammonium ion [1], the decrease of pI value of enzymatically methylated cytochrome c, as demonstrated in Fig. 1, was quite unexpected. Thus, in order to further investigate the effect of proteinlysine methylation on the structure of cytochrome c, we attempted to construct a space-filling model of both unmethylated and methylated horse heart cytochrome c. However, we soon realized that this would be prohibitively expensive. Therefore, we built models of undecapeptide of residues 70--80. This undecapeptide is the longest peptide in the cytochrome c which has been shown to be invariant during evolution and also contains residue-72 lysine [16]. At first sight, both models of unmethylated (Fig. 2A) and methylated (Fig. 2B) undecapeptide do not seem to be significantly different from each other in their appearances. However, close examination reveals that unmethylated undecapeptide (Fig. 3A) has a hydrogen bond (shown by arrow and flat block) between the oxygen of amide of residue-70 asDaragine and the nitrogen of
Fig. 2. Space-filling m o d e l s o f u n d e c a p e p t i d e o f residues 7 0 - - 8 0 . T h e u n d e c a p e p t i d e is c o m p o s e d o f Asn-Pro-Lys-Lys-Tyr-Ile-Pro-Gly-Thr-Lys-Met. A, u n m e t h y l a t e d and a r r o w i n d i c a t e s t h e h y d r o g e n b o n d 70 75 80 b e t w e e n t h e o x y g e n o f a m i d e o f r e s i d u e - 7 0 asp~ragine a n d t h e n i t r o g e n o f residue-72 lysine. B, m e t h y l ated.
149
Fig. 3. Close-up of m o d e l s presented in Fig. 2. A, u n m e t h y l a t e d . B, m e t h y l a t e d .
e-amino group of residue-72 lysine. This hydrogen bond tends to dissociate when the e-amino group is substituted with three methyl groups (Fig. 3B). Discussion
Substitution of the e-amino group of lysine with three methyl groups makes the nitrogen quaternary ammonium ion, thus rendering it permanently positively charged. This therefore would raise the pI of methylated eytoehrome e to a higher scale. Thus, it was a great surprise to observe that the pI value of enzymatieally methylated eytoehrome e was actually 0.54 unit lower than that of unmethylated eytoehrome e. This decrease of pI value is not consistent with the introduction of a fixed positive charge into the e-amino group of residue-72 lysine. The results suggest a more gross effect in that the 'effective charge' of the entire molecule is lowered. Specifically, enzymatic trimethylation of eytoehrome e interferes with the hydrogen bond between the oxygen of the amide of residue-70 asparagine and the nitrogen of residue-72 lysine. We therefore propose that, although the residue-72 obtained a fixed positive charge, the enzymatic trimethylation of eytoehrome e also results in the redistribution of charges, thereby rendering the protein molecule less basic. The primary event in bringing the change in the tertiary structure of the cytochrome c might be the breakage of hydrogen bond between the residue-70 asparagine and residue-72 lysine. The above result also explains the 'anomalous' elution pattern of an unmethylated cytochrome c from CM-cellulose or Bio-Rex 70 resin observed by others [10,17]; methylated cytochrome c is effectively less basic than the unmethylated species. Finally, it should be pointed out that this is the first instance where the difference of charge in the methylated protein has ever been demonstrated.
150
Acknowledgements This work was supported by research grants AM09602 from the National Institute of Arthritis, Metabolism, and Digestive Diseases, CA10439 and CA12226 from the National Cancer Institute, and GM20594 from the National Institute of General Medical Sciences. References I Paik, W.K. and Kim, S. (1971) Science 174, 114--119 2 Paik, W.K. and Kim, S. (1975) in Advances in E n z y m o l o g y (Meister, A., ed.), Vol. 42, pp. 227--286, John Wiley an d Sons, New Y o r k 3 DeLange, R.J., Glazer, A.N. and Smith, E.L. (1969) J. Biol. Chem. 244, 1385--1388 4 Nochumson, S., Durban, E., Kim, S. and Paik, W.K. (1977) Biochem. J. 165, 11--18 5 Durban, E., Nochumson, S., Kim, S., Paik, W.K. and Chart, S.-K. (1978) J. Biol. Chem. 253, 1427--1435 6 DiMarla, P., Polastro, E., DeLange, R.J., Kim, S. and Paik, W.K. (1979) J. Biol. Chem. 254, 4645--4652 7 Po]astro, E, Deconinck, M.M., Devogel, M . R , Maillier, E.L., Looze, Y., Schneck, A.C. and Leonis, J. (1978) F E B S Lett. 86, 17--24 8 Polastro, E., Schneck, A.G, Leonis, J., Kim, S. and Paik, W.K. (1978) Int. J. Biochem. 9,795--801 9 Margoliash, E. and Walasek, O.F. (1967) Methods Enzymol. 10,339--348 10 Scott, W.A. and Mitchell, H.K. (1969) Biochemistry 8, 4 2 8 2 - 4 2 8 9 11 Vesterberg, O. (1971) Methods Enzymol. 22, 389--412 12 Paik, W.K. and Kim, S. (1967) Biochem. Biophys. Res. Commun. 27, 479--483 13 Flatmark , T. and Vesterberg, O. (1966) Acta Chem. Scand. 20, 1497--1503 14 O'Fa~Tell, P.Z., Go odman, H.M. and O'FaITell, P.H. (1977) Cell 12, 1 1 3 3 - - 1 1 4 2 15 VeSterberg, O. and Svenson, H. (1966) Acta Chem. Scand. 20, 820--834 16 MargoHash, E. and Scheiter, A. (1966) in Advances in Protein Chemistry (Anfinsen, C.B., Anson, M.L., Edsall, J.T. and Richards, F.H., eds.), Vol. 21, pp. 113--286, Academic Press, New Y ork 17 Foucher, M., Verdiere, J., Lederer, F. and Slonimski, P. (1972) Eu~. J. Biochem. 3 1 , 1 3 9 - - 1 4 3 18 Theorell, H. and Aekesson, A. (1941) J, Am. Chem. Soc. 63, 1804--1811 19 Tint, H. and Reiss, W. (1950) J. Biol. Chem. 1 8 2 , 3 8 5 - - 3 9 6 20 Shirasaka, M., Nak ayama, N., Endo, A., Haneishi, T. and Akazaki, H. (1968) J. Biochem. (Tokyo) 63, 417-424 21 Neilands, J.B. (1952) J. Biol. Chem. 197, 701--708 22 Motonaga, K., Misaka, E., Nakajima, E., Ueda, S. and Nakanishi, K. (1965) J. Biochem. (Tokyo) 57, 22--28 23 Heller, J. and Smith, E.L. (1966) J. Biol. Chem. 241, 3 1 5 8 - - 3 1 6 4
Biochimica et Biophysica Acta, 6 2 2 ( 1 9 8 0 ) 1 4 4 - - 1 5 0 © E l s e v i e r / N o r t h - H o l l a n d B i o m e d i c a l Press
BBA 38382
ENZYMATIC TRIMETHYLATION OF RESIDUE-72 LYSINE IN CYTOCHROME c EFFECT ON THE TOTAL STRUCTURE
C H A N G - S E KIM *, F. K U E P P E R S **, P. D I M A R I A , J. F A R O O Q U I , S. KIM a n d W.K. P A I K
Fels Research Institute and Department of Biochemistry, Temple University School of Medicine, Philadelphia, PA 19140 (U.S.A.) (Received June 27th, 1979)
Key words: Cytochrome c methylation; Isoelectric focusing; Space-filling model; Protein methyluse; (N. crassa, S. cerevisiae)
Summary A highly purified protein methylase III from Neurospora crassa or Saccharom y c es cerevisiae specifically methylates a single lysine residue of position 72 of horse heart cytochrome c. The enzymatically methylated cytochrome c has been separated from the unmethylated counterpart species by isoelectric focusing. Simultaneously, the pI values of these two species were found to be 9.49 and 10.03, respectively. Since methyl substitution increases the basicity associated with the e-amino group of lysine residues, the observed decrease in pI value is in opposition to the predicted increase. Space-filling models revealed the possibility of a hydrogen bond between the oxygen of amide of residue-70 asparagine and the e-amino nitrogen of residue-72 lysine in unmethylated horse heart cytochrome c. The enzymatic methylation of residue-72 lysine tends to dissociate this hydrogen bond, thereby possibly inducing the shift of 'effective charge' of the protein molecule. This paper also deals with the pI values of cytochromes c from 13 different sources, determined by the isoelectric focusing technique.
* Present address: D e p a r t m e n t of Biochemistry, Chosun University School of Medicine, Kwangju, Korea. ** Present address: Department o f Medicine, Temple University School of Medicine, Philadelphia, PA 19140, U.S.A.
145 Introduction
Protein methylation is one of the postt~anslational modification reactions, which occurs ubiquitously in many proteins involving various amino acid side chains [1,2]. In particular, DeLange et al. [3] earlier observed that cytochrome c of lower organisms have the residue-72 lysine methylated in vivo. In accordance with this observation, we have recently identified an enzyme from Neurospora crassa and Saccharomyces cerevisiae which methylates only a single lysine of position 72 of in vivo unmethylated horse cytochrome c [4--6]. The enzymatic methylation of cytochrome c facilitates the binding of this protein to mitochondria [ 7,8]. In an attempt to further understand the mechanism of the facilitation of cytochrome c binding to mitochondria by lysine methylation, the determination of the pI values of enzymatically methylated and unmethylated cytochrome c by isoelectric focusing was performed: the pI values of these two species were found to be 9.49 and 10.03, respectively. Attempts were also being made to build a space-filling model of residues 70--80 which is invariant among the various cytochromes c throughout evolution. Materials and Methods Materials. Cytochromes c of various species: horse heart, S. cerevisiae, bovine heart, pigeon heart, rabbit heart, tuna heart, Candida krusei, chicken heart, porcine heart and canine heart were obtained from Sigma Chemical Co. Cytochromes c of guanaco and mouse heart were prepared according to the method of Margoliash and Walasek [9], and N. crassa by the method of Scott and Mitchell [ 10]. S-Adenyl-L-[Me-14C]methionine (specific activity, 54.7 Ci/mol) and Ampholine (pH 9--11) were obtained from New England Nuclear and LKB, Stockholm, respectively. All other reagents were of the highest grade obtainable. Preparation o f [Me-~4C]cytochrome c. Enzymatically methylated horse heart cytochrome c was prepared according to the published method [5]. The reaction mixture contained 0.1 M glycine/NaOH buffer (pH 9.0), 5.7 mM mercaptoethanol, 0.4 mM EDTA, 30 mg horse heart cytochrome c, 40 pM S-adenosylL-[Me-~4C]methionine (100 cpm/pmol) and 20 pg of highly purified protein methylase III of N. crassa (S-adenosylmethionine:protein(lysine)N-methyltransferase, EC 2.1.1.43) in a final total volume of 1.2 ml. After 1 h incubation at 37°C, an additional 0.2 ml (20 pg) of enzyme was added, and the reaction was allowed to proceed for another 1 h. In order to remove unreacted S-adenosyl-L-[Me-laC]methionine, the reaction mixture was dialyzed against deionized water for 6 h with occasional changes. The sample was lyophilized until use.
Isoelectric focusing. Isoelectric focusing of cytochrome c from various species was performed according to the method of Vesterberg [11], usinga 110 ml column. A linear gradient was prepared by mixing light and dense solution. The light solution was made up of 59.5 ml of water and 0.5 ml of LKB Ampholine (pH 9--11, 20%). The dense solution was comprised of 28 g of sorbitol dissolved in 38.5 ml of water and 1.5 ml of Ampholine (pH 9--11, 20%). The gra-
146 dients were laid on t o p of the dense anode solution which was prepared by dissolving 12 g of sorbitol in 14 ml of water containing 0.1 ml of concentrated sulfuric acid. The cathode solution containing 0.05 g of NaOH was carefully layered on top of the sorbitol gradients. Isoelectric focusing was carried o u t in a water-jacket LKB 8100 column at 0--2°C and 800 V for 20--24 h. The column was then eluted from the b o t t o m using a Buchler multistaltic pump with a flow rate of 0.4 ml/min. Fractions of 2 ml were collected and the pH and Ass0 of each fractions were measured at room temperature. Radioactivity in each fraction was measured in an aliquot of 0.1 ml using scintillation Formula 963 (New England Nuclear Corp.). Amino acid analysis. Proteins were hydrolyzed in 6 N HC1 for 48 h at l l 0 ° C in vacuo. Amino acid analysis was carried o u t as described by Paik and Kim [12]. Results
Separation of enzymatically methylated [Me-14C]cytochrome c of horse heart from unmethylated species by isoelectric focusing Fig. I illustrates the separation of enzymatically methylated horse heart c y t o c h r o m e c from unmethylated species by isoelectric focusing. It had previously been demonstrated in this laboratory that c y t o c h r o m e c-specific protein methylase III of N. crassa methylated a single lysine residue at position 72 among 19 lysine residues present in c y t o c h r o m e c molecule and that the
10.03
°"
10.~ 10.(
400
~H ~.~" 9.¢ ~.~ ] cpm
cpm
o.(
~A~o
i o., Asso
0.2
,~ ~'~ ~
~'o ~
s6 ~
Fraction N o . focusing of enzyrnaticaliy methylated [Me-14C]cytoch~ome c o f h o r s e hesxt. Horse c was methylated i n v i t r o b y a h i g h l y p u r i f i e d N. crassa p r o t e i n m e t h y l a s e I I I w i t h S-adenosyl-L-[Me-14C]methionine. A f t e r d i a l y s i s , t]~e s a m p l e w a s a n a l y z e d o n a n e l e c t r o f o c u s i n g w i t h A m p h o H n e of pH 9--11. Methylated cytochrome c is indicated by radioactivity, and tmmethy]ated (cont r o l ) c y t o c h r o m e c b y A 5 50 • F i g . 1. I s o e l e c t r i c heart cytoch~ome
147
enzymatic methylation in vitro proceeded to a maximum of 0.5% of the total cytochrome c population [4,5]. Thus, when separated by isoelectric focusing, the methylated cytochrome c can only be visualized by its radioactivity (Fig. 1), and the unmethylated cytochrome c by the absorbance at 550 nm. As shown in Fig. 1, the major bands of radioactivity and A55onm are clearly separated by isoelectric focusing, using Ampholine pH 9--11. The isoelectric points (pI) of both methylated and unmethylated horse heart cytochromes c were determined to be 9.49 and 10.03, respectively. In order to confirm that radioactive peak is indeed methylated cytochrome c, the peak radioactivity fractions (fractions 33--35) were pooled, dialyzed and lyophilized. Amino acid analysis of the sample indicated that the radioactivity represented mostly trimethyllysine. The presence of trimethyllysine further ruled out the possibility of this peak as deamidated cytochrome c observed by Flatmark and Vesterberg in the case of beef heart cytochrome c [13]. The separation of methylated cytochrome c from unmethylated was also done using isoelectric focusing on polyacrylamide gels as described by O'Farrell et al. [14]. The pattern of separation was the same as that obtained with the preparative method (not shown). p I values o f cytochromes c from various sources Table I lists pI values of cytochrome c isolated from various sources determined by us as well as corresponding values reported in the literature. In comparing our values with the corresponding reported values, it is seen that with the exception of C. krusei and N. crassa cytochromes c our values are slightly lower. This can be attributed to the fact that our values were determined at TABLE I ISOELECTRIC POINTS (pl) OF CYTOCHROMES
c ISOLATED FROM VARIOUS SOURCES
T h e n u m b e r s in b r a c k e t s i n d i c a t e r e f e r e n c e n u m b e r s . F o r p r e s e n t w o r k p I values w e r e d e t e r m i n e d at 2 4 -+ I ° C . F o r o t h e r s ' w o r k p I values w e r e d e t e r m i n e d at 4 ° C . Sources of cytochrome c
Isoelectric point (pl) Present work
Others' w o r k
Horse h e a r t
10.03
10.65 10.9 10.35 10.7
[18] [19] [20] [21]
Mouse heart Rabbit heart Bovine heart Guanaco heart Tuna heart Canine h e a r t Porcine heart Chicken heart P i g e o n heart C. k r u s e i $. cerevisiae N. crassa Horse heart; enzymatically methylated
10.03 10.02 10.01 10.01 10.01 10.01 10.00 10.00 9.98 9.98 9.91 9.40 9.49
10.8 10.8
[19] [19]
9.8 [20] 10.4--10.5 [22] 9.34 [23]
* I t w a s f o u n d ~hat, w i t h all p r e p a r a t i o n s , t h e r e w e r e m o r e t h a n o n e b a n d i n d i c a t i n g c o n t a m i n a t i o n . T h u s , p I values o f m a j o r b a n d have b e e n t a k e n .
148 25°C as opposed to 4°C for the values determined by others (it is known that pI measurement is dependent on temperature [15]}. Looking at the cytochromes c of higher organisms, it can be seen th~t their pI values are close to 10 or little higher. The absence of in vivo methylated lysine residues have been reported in these cytochrome c species [3]. Only N. crassa and in vitro enzymatically methylated horse heart cytochromes c have pI values significantly below those of the higher organisms. Space-filling model It has previously been shown that e-N-trimethyllys.ine is the predominant product of in vitro enzymatically methylated cytochrome c along with the presence of small amounts of e-N-mono- and e-N-dimethyllysine [4,5]. Since the introduction of three methyl groups to the e-amino group of lysine residues makes the nitrogen a quaternary ammonium ion [1], the decrease of pI value of enzymatically methylated cytochrome c, as demonstrated in Fig. 1, was quite unexpected. Thus, in order to further investigate the effect of proteinlysine methylation on the structure of cytochrome c, we attempted to construct a space-filling model of both unmethylated and methylated horse heart cytochrome c. However, we soon realized that this would be prohibitively expensive. Therefore, we built models of undecapeptide of residues 70--80. This undecapeptide is the longest peptide in the cytochrome c which has been shown to be invariant during evolution and also contains residue-72 lysine [16]. At first sight, both models of unmethylated (Fig. 2A) and methylated (Fig. 2B) undecapeptide do not seem to be significantly different from each other in their appearances. However, close examination reveals that unmethylated undecapeptide (Fig. 3A) has a hydrogen bond (shown by arrow and flat block) between the oxygen of amide of residue-70 asDaragine and the nitrogen of
Fig. 2. Space-filling m o d e l s o f u n d e c a p e p t i d e o f residues 7 0 - - 8 0 . T h e u n d e c a p e p t i d e is c o m p o s e d o f Asn-Pro-Lys-Lys-Tyr-Ile-Pro-Gly-Thr-Lys-Met. A, u n m e t h y l a t e d and a r r o w i n d i c a t e s t h e h y d r o g e n b o n d 70 75 80 b e t w e e n t h e o x y g e n o f a m i d e o f r e s i d u e - 7 0 asp~ragine a n d t h e n i t r o g e n o f residue-72 lysine. B, m e t h y l ated.
149
Fig. 3. Close-up of m o d e l s presented in Fig. 2. A, u n m e t h y l a t e d . B, m e t h y l a t e d .
e-amino group of residue-72 lysine. This hydrogen bond tends to dissociate when the e-amino group is substituted with three methyl groups (Fig. 3B). Discussion
Substitution of the e-amino group of lysine with three methyl groups makes the nitrogen quaternary ammonium ion, thus rendering it permanently positively charged. This therefore would raise the pI of methylated eytoehrome e to a higher scale. Thus, it was a great surprise to observe that the pI value of enzymatieally methylated eytoehrome e was actually 0.54 unit lower than that of unmethylated eytoehrome e. This decrease of pI value is not consistent with the introduction of a fixed positive charge into the e-amino group of residue-72 lysine. The results suggest a more gross effect in that the 'effective charge' of the entire molecule is lowered. Specifically, enzymatic trimethylation of eytoehrome e interferes with the hydrogen bond between the oxygen of the amide of residue-70 asparagine and the nitrogen of residue-72 lysine. We therefore propose that, although the residue-72 obtained a fixed positive charge, the enzymatic trimethylation of eytoehrome e also results in the redistribution of charges, thereby rendering the protein molecule less basic. The primary event in bringing the change in the tertiary structure of the cytochrome c might be the breakage of hydrogen bond between the residue-70 asparagine and residue-72 lysine. The above result also explains the 'anomalous' elution pattern of an unmethylated cytochrome c from CM-cellulose or Bio-Rex 70 resin observed by others [10,17]; methylated cytochrome c is effectively less basic than the unmethylated species. Finally, it should be pointed out that this is the first instance where the difference of charge in the methylated protein has ever been demonstrated.
150
Acknowledgements This work was supported by research grants AM09602 from the National Institute of Arthritis, Metabolism, and Digestive Diseases, CA10439 and CA12226 from the National Cancer Institute, and GM20594 from the National Institute of General Medical Sciences. References I Paik, W.K. and Kim, S. (1971) Science 174, 114--119 2 Paik, W.K. and Kim, S. (1975) in Advances in E n z y m o l o g y (Meister, A., ed.), Vol. 42, pp. 227--286, John Wiley an d Sons, New Y o r k 3 DeLange, R.J., Glazer, A.N. and Smith, E.L. (1969) J. Biol. Chem. 244, 1385--1388 4 Nochumson, S., Durban, E., Kim, S. and Paik, W.K. (1977) Biochem. J. 165, 11--18 5 Durban, E., Nochumson, S., Kim, S., Paik, W.K. and Chart, S.-K. (1978) J. Biol. Chem. 253, 1427--1435 6 DiMarla, P., Polastro, E., DeLange, R.J., Kim, S. and Paik, W.K. (1979) J. Biol. Chem. 254, 4645--4652 7 Po]astro, E, Deconinck, M.M., Devogel, M . R , Maillier, E.L., Looze, Y., Schneck, A.C. and Leonis, J. (1978) F E B S Lett. 86, 17--24 8 Polastro, E., Schneck, A.G, Leonis, J., Kim, S. and Paik, W.K. (1978) Int. J. Biochem. 9,795--801 9 Margoliash, E. and Walasek, O.F. (1967) Methods Enzymol. 10,339--348 10 Scott, W.A. and Mitchell, H.K. (1969) Biochemistry 8, 4 2 8 2 - 4 2 8 9 11 Vesterberg, O. (1971) Methods Enzymol. 22, 389--412 12 Paik, W.K. and Kim, S. (1967) Biochem. Biophys. Res. Commun. 27, 479--483 13 Flatmark , T. and Vesterberg, O. (1966) Acta Chem. Scand. 20, 1497--1503 14 O'Fa~Tell, P.Z., Go odman, H.M. and O'FaITell, P.H. (1977) Cell 12, 1 1 3 3 - - 1 1 4 2 15 VeSterberg, O. and Svenson, H. (1966) Acta Chem. Scand. 20, 820--834 16 MargoHash, E. and Scheiter, A. (1966) in Advances in Protein Chemistry (Anfinsen, C.B., Anson, M.L., Edsall, J.T. and Richards, F.H., eds.), Vol. 21, pp. 113--286, Academic Press, New Y ork 17 Foucher, M., Verdiere, J., Lederer, F. and Slonimski, P. (1972) Eu~. J. Biochem. 3 1 , 1 3 9 - - 1 4 3 18 Theorell, H. and Aekesson, A. (1941) J, Am. Chem. Soc. 63, 1804--1811 19 Tint, H. and Reiss, W. (1950) J. Biol. Chem. 1 8 2 , 3 8 5 - - 3 9 6 20 Shirasaka, M., Nak ayama, N., Endo, A., Haneishi, T. and Akazaki, H. (1968) J. Biochem. (Tokyo) 63, 417-424 21 Neilands, J.B. (1952) J. Biol. Chem. 197, 701--708 22 Motonaga, K., Misaka, E., Nakajima, E., Ueda, S. and Nakanishi, K. (1965) J. Biochem. (Tokyo) 57, 22--28 23 Heller, J. and Smith, E.L. (1966) J. Biol. Chem. 241, 3 1 5 8 - - 3 1 6 4