Physicochemical properties of two atypical cytochromes c, Crithidia cytochrome c-557 and Euglena cytochrome c-558.

Cytochrome c-557 from Crithidia oncopelti and cytochrome c-558 from Euglena gracilis are mitochondrial cytochromes c that have an atypical haem-binding site. It was of interest to know whether the loss of one thioether bond affected the physicochemical properties of these cytochromes. The thermodynamic parameters of the redox potential were measured. The reaction with imidazole, the kinetics and thermodynamics of the alkaline isomerization and the effect of heating on the visible spectrum are described for the ferricytochromes. The kinetics of the loss of cyanide, the spectral changes occurring on reduction with dithionite at alkaline pH values and the reactivity with CO are described for the ferrocytochromes. In many respects the cytochromes of the two protozoans are very similar to the cytochromes of horse and yeast. The ferricytochromes do, however, undergo a reversible transition to high-spin species on heating, which may be due to the more flexible attachment of the prosthetic group. Similarly the alkaline isomers of cytochromes c-557 and c-558 give rise to high-spin proteins above pH 11. The alkaline isomerization of cytochrome c-558, involves a pKobs. of 10 and kinetics which do not obey the model of Davis et al. [(1974) J. Biol. Chem. 249, 2624-2632] for horse cytochrome c. It is proposed that a model involving two ionizations, followed by a conformation change, may fit the data. Both cytochromes c-557 and c-558 combine slowly with CO at neutral pH values.     

Enzymatic methyl esterification of synthetic tripeptides: structural requirements of the peptide substrate. Detection of the reaction products by fast-atom-bombardment mass spectrometry

Eukaryotic protein carboxyl methyltransferase catalyzes a two-substrates reaction in which the methyl group of S-adenosylmethionine is transferred to the free carboxyl group of D-aspartyl and L-isoaspartyl-containing peptide or protein substrates. It has been previously shown that at least three binding sites are required for the interaction of adenosylmethionine with the enzyme and/or the protein substrate [Oliva A., Galletti P., Zappia V., Paik W. K. & Kim S. (1980) Eur. J. Biochem. 104, 595-602], while very little is known concerning the structural requirements of the protein substrate. In this study several synthetic tripeptides were selected in order to elucidate the structural requirements of the methyl-accepting substrates. The results obtained with this series of peptides suggested that: (1) three residues appear to be the minimal length, so far identified, required for a productive enzyme-substrate interaction, several dipeptides being ineffective as substrates [McFadden P. N. & Clarke S. (1986) J. Biol. Chem. 261, 11,503-11,511]; (2) the isoaspartyl residue is not recognized unless its alpha-amino group is involved in a carboamide bond; (3) an hydrogen atom on the amide linkage following the isoaspartyl residue is essential for both recognition and catalysis; (4) oligopeptides containing both D-aspartyl and D-isoaspartyl residues are not recognized by this methyltransferase. On the basis of these results, interaction sites between the peptide substrate and the enzyme molecule have been proposed. This paper also reports the first application of fast-atom-bombardment mass spectrometry to the detection of the products of the enzymatic methyl esterification reaction. By this soft ionization technique, the methyl-esterified peptides as well as the corresponding cyclic imides generated during the spontaneous demethylation process have been identified.     

Biosynthesis of diphthamide in Saccharomyces cerevisiae. Partial purification and characterization of a specific S-adenosylmethionine:elongation factor 2 methyltransferase

The inactivation of elongation factor 2 (EF-2) by diphtheria toxin requires the presence of a post-translationally modified histidine residue in EF-2. This residue, diphthamide, has the structure 2-[3-carboxyamido-3-(trimethylammonio)propyl]histidine. The present work was undertaken to study the pathway of diphthamide biosynthesis using diphtheria toxin-resistant yeast mutants (Chen. J.-Y., Bodley, J. W., and Livingston, D. M. (1985) Mol. Cell. Biol. 5, 3357-3360) which are defective in diphthamide formation. We demonstrate here that one of these mutants (dph5) contains a toxin-resistant form of EF-2 which can be converted in vitro to a toxin-sensitive form through the action of an enzyme present in other yeast strains. Both this toxin-resistant EF-2 and its modifying enzyme have been partially purified and evidence is presented that the modifying enzyme is a specific S-adenosylmethionine:EF-2 methyltransferase. In vitro complementation to diphtheria toxin sensitivity required S-adenosylmethionine, and when partially purified components were incubated with [methyl-3H]S-adenosylmethionine, label was incorporated specifically into EF-2. Hydrolysis of labeled EF-2 yielded diphthine (the unamidated form of diphthamide) and a single chromatographically separable labeling intermediate. We conclude that the S-adenosylmethionine:EF-2 methyltransferase adds at least the last two of the three methyl groups present in diphthine and that this modification is sufficient to create diphtheria toxin sensitivity. Evidence is also presented for the existence of an ATP-dependent amidating enzyme which catalyzes the final step in the biosynthesis of diphthamide in EF-2.     

Sulfite oxidase from chicken liver. The role of imidazole and carboxyl groups for the reaction with cytochrome c

Oxidation of sulfite to sulfate by sulfite oxidase is inhibited when the enzyme is treated with reagents known to modify imidazole and carboxyl groups. Modification inhibits the oxidation of sulfite by the physiological electron acceptor cytochrome c, but not by the artificial acceptor ferricyanide. This indicates interference with reaction steps that follow the oxidation of sulfite by the enzyme's molybdenum cofactor. Reaction with diethylpyrocarbonate modifies ten histidines per enzyme monomer. Loss of activity is concomitant to the modification of only a single histidine residue. Inactivation takes place at the same rate in free sulfite oxidase and in the sulfite-oxidase--cytochrome-c complex. Blocking of carboxyl groups with water-soluble carbodiimides inactivates the enzyme. But none of the enzyme's carboxyl groups seems to be essential in the sense that its modification fully abolishes activity. The pattern of inactivation by chemical modification of sulfite oxidase is quite similar to that observed previously for cytochrome c peroxidase from yeast [Bosshard, H. R., B?nziger, J., Hasler, T. and Poulos, T. L. (1984) J. Biol. Chem. 259, 5683-5690; Bechtold, R. and Bosshard, H. R. (1985) J. Biol. Chem. 260, 5191-5200]. The two enzymes have very different structures yet share cytochrome c as a common substrate of which they recognize the same electron-transfer domain around the exposed heme edge.     

Enzymatic methylation of band 3 anion transporter in intact human erythrocytes

Band 3, the anion transport protein of erythrocyte membranes, is a major methyl-accepting substrate of the intracellular erythrocyte protein carboxyl methyltransferase (S-adenosyl-L-methionine: protein-D-aspartate O-methyltransferase; EC 2.1.1.77) [Freitag, C., & Clarke, S. (1981) J. Biol. Chem. 256, 6102-6108]. The localization of methylation sites in intact cells by analysis of proteolytic fragments indicated that sites were present in the cytoplasmic N-terminal domain as well as the membranous C-terminal portion of the polypeptide. The amino acid residues that serve as carboxyl methylation sites of the erythrocyte anion transporter were also investigated. 3H-Methylated band 3 was purified from intact erythrocytes incubated with L-[methyl-3H]methionine and from trypsinized and lysed erythrocytes incubated with S-adenosyl-L-[methyl-3H]methionine. After proteolytic digestion with carboxypeptidase Y, D-aspartic acid beta-[3H]methyl ester was isolated in low yields (9% and 1%, respectively) from each preparation. The bulk of the radioactivity was recovered as [3H]methanol, and the amino acid residue(s) originally associated with these methyl groups could not be determined. No L-aspartic acid beta-[3H]methyl ester or glutamyl gamma-[3H]methyl ester was detected. The formation of D-aspartic acid beta-[3H]methyl esters in this protein in intact cells resulted from protein carboxyl methyltransferase activity since it was inhibited by adenosine and homocysteine thiolactone, which increases the intracellular concentration of the potent product inhibitor S-adenosylhomocysteine, and cycloleucine, which prevents the formation of the substrate S-adenosyl-L-[methyl-3H]methionine.     

Purification of S-adenosylmethionine: epsilon-N-L-lysine methyltransferase. The first enzyme in carnitine biosynthesis.

The initial steps of carnitine biosynthesis in Neurospora crassa involve the methylation of the epsilon-amino group of lysine as follows: Lysine A leads to monomethyllysine B leads to dimethyllysine C leads to trimethyllysine. The methyl donor is S-adenosylmethionine. An enzyme, S-adenosylmethionine:epsilon-N-L-lysine methyltransferase, has been purified from N. crassa to near homogeneity as judged by column chromatography, polyacrylamide gel electrophoresis, and ultracentrifugation. This protein catalyzes all three methylation reactions. The reaction rates are: A less than B less than C. Sedimentation equilibrium and molecular filtration give a molecular weight of 22,000 for the protein. Sedimentation equilibrium analysis of the protein in 6 M guanidine hydrochloride and sodium dodecyl sulfate-polyacrylamide gel electrophoresis do not detect the possibility of subunit structure. The enzyme contains no half-cystine but does contain several acidic residues. The protein exhibits an absorption band between 400 and 420 nm which is 40 to 50 times less than the absorption seen at 280 nm and is not affected by the presence of substrates. The source of this absorption in unkown.     

Mechanism of multiple lysine methylation by the SET domain enzyme Rubisco LSMT

SET domain protein methyltransferases catalyze the transfer of methyl groups from the cofactor S-adenosylmethionine (AdoMet) to specific lysine residues of protein substrates, such as the N-terminal tails of histones H3 and H4 and the large subunit of the Rubisco holoenzyme complex. The crystal structures of pea Rubisco large subunit methyltransferase (LSMT) in ternary complexes with either lysine or epsilon-N-methyllysine (MeLys) and the product S-adenosylhomocysteine (AdoHcy) were determined to resolutions of 2.65 and 2.55 A, respectively. The zeta-methyl group of MeLys is bound to the enzyme via carbon-oxygen hydrogen bonds that play a key role in catalysis. The methyl donor and acceptor are aligned in a linear geometry for S(N)2 nucleophilic transfer of the methyl group during catalysis. Differences in hydrogen bonding between the MeLys epsilon-amino group and Rubisco LSMT and SET7/9 explain why Rubisco LSMT generates multiply methylated Lys, wheras SET7/9 generates only MeLys.     

Effect of the hinge protein on the heme iron site of cytochrome c1

X-ray absorption spectroscopic (XAS) studies on cytochrome C1 from beef heart mitochondria were conducted to identify the effect of the hinge protein [Kim, C.H., & King, T.E. (1983) J. Biol. Chem. 258, 13543-13551] on the structure of the heme site in cytochrome c1. A comparison of XAS data of highly purified "one-band" and "two-band" cytochrome c1 [Kim, C.H., & King, T.E. (1987) Biochemistry 26, 1955-1961] demonstrates that the hinge protein exerts a rather pronounced effect on the heme environment of the cytochrome c1: a conformational change occurs within a radius of approximately 5 A from the heme iron in cytochrome c1 when the hinge protein is bound to cytochrome c1. This result may be correlated with the previous observations that the structure and reactivity of cytochrome c1 are affected by the hinge protein [Kim, C.H., & King, T.E. (1987) Biochemistry 26, 1955-1961; Kim, C.H., Balny, C., & King, T.E. (1987) J. Biol. Chem. 262, 8103-8108].     

Methylation of calmodulin at carboxylic acid residues in erythrocytes. A non-regulatory covalent modification?

The physiological role of protein carboxy-group methylation reactions in human erythrocytes was studied with calmodulin as an endogenous methyl-group acceptor. The steady-state degree of calmodulin carboxy-group methylation is substoichiometric both in intact cells and in a lysed-cell system (about 0.0003 mol of methyl groups/mol of polypeptide). Purified erythrocyte calmodulin is a substrate for a partially purified erythrocyte carboxy-group methyltransferase and can be methylated to the extent of about 0.0007-0.001 mol of methyl groups/mol of polypeptide. This erythrocyte protein methyltransferase displays an apparent specificity for atypical racemized and/or isomerized D-aspartate and L-isoaspartate residues [McFadden & Clarke (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 2460-2464; Murray & Clarke (1984) J. Biol. Chem. 259, 10722-10732]. Exposure of calmodulin to elevated temperatures before methylation results in racemization of aspartate and/or asparagine residues, and may result in isoaspartate formation as well. The methylatability of these samples also increases as a function of time of heating, independent of the pH (over the range pH 5-9) or Ca2+ concentration; the most significant increase occurs during the initial 60 min, when calmodulin retains a fraction of its biological activity. These results are consistent with the hypothesis that methylation of calmodulin may occur at these uncommon aspartate residues, but are not consistent with a regulatory role for the methylation reaction.     

Effects of a methyl-deficient diet on rat liver phosphatidylcholine biosynthesis

To produce a severe choline-methionine deficiency, a synthetic L-amino acid diet, free of choline, methionine, vitamin B12, and folic acid and supplemented with guanidoacetic acid, a methyl group acceptor, was fed to female rats for 2 weeks. The in vitro activity of liver microsomal phosphatidylethanolamine methyltransferase was stimulated twofold when compared with basal diet controls. The activity of choline phosphotransferase was depressed by 86%; thus, the contribution of the methyltransferase in the overall synthesis of phosphatidylcholine apparently increased. However, measurement of the in vivo methylation of phosphatidylethanolamine by incorporation of [1,2-14C]ethanolamine into phosphatidylcholine indicates that the methylation pathway is markedly depressed in methyl deficiency. Hepatic concentrations of the methyltransferase substrate, S-adenosylmethionine, and the inhibitory metabolite, S-adenosylhomocysteine, were significantly altered such that an unfavorable environment for methylation was present in the deficient animal. The ratio of substrate to inhibitor was depressed from 5.2:1 in the controls to 1.7:1 in the livers of methyl-depleted rats. Control of transmethylation in accordance with the availability of substrates, phosphatidylethanolamine, or S-adenosylmethionine, and the level of S-adenosylhomocysteine is discussed.     

Methylation of H3-lysine 79 is mediated by a new family of HMTases without a SET domain

The N-terminal tails of core histones are subjected to multiple covalent modifications, including acetylation, methylation, and phosphorylation. Similar to acetylation, histone methylation has emerged as an important player in regulating chromatin dynamics and gene activity. Histone methylation occurs on arginine and lysine residues and is catalyzed by two families of proteins, the protein arginine methyltransferase family and the SET-domain-containing methyltransferase family. Here, we report that lysine 79 (K79) of H3, located in the globular domain, can be methylated. K79 methylation occurs in a variety of organisms ranging from yeast to human. In budding yeast, K79 methylation is mediated by the silencing protein DOT1. Consistent with conservation of K79 methylation, DOT1 homologs can be found in a variety of eukaryotic organisms. We identified a human DOT1-like (DOT1L) protein and demonstrated that this protein possesses intrinsic H3-K79-specific histone methyltransferase (HMTase) activity in vitro and in vivo. Furthermore, we found that K79 methylation level is regulated throughout the cell cycle. Thus, our studies reveal a new methylation site and define a novel family of histone lysine methyltransferase.     

Hsl7p, the yeast homologue of human JBP1, is a protein methyltransferase

The yeast protein Hsl7p is a homologue of Janus kinase binding protein 1, JBP1, a newly characterized protein methyltransferase. In this report, Hsl7p also is shown to be a methyltransferase. It can be crosslinked to [(3)H]S-adenosylmethionine and exhibits in vitro protein methylation activity. Calf histones H2A and H4 and bovine myelin basic protein were methylated by Hsl7p, whereas histones H1, H2B, and H3 and bovine cytochrome c were not. We demonstrated that JBP1 can complement Saccharomyces cerevisiae with a disrupted HSL7 gene as judged by a reduction of the elongated bud phenotype, and a point mutation in the JBP1 S-adenosylmethionine consensus binding sequence eliminated all complementation by JBP1. Therefore, we conclude the yeast protein Hsl7p is a sequence and functional homologue of JBP1. These data provide evidence for an intricate link between protein methylation and macroscopic changes in yeast morphology.     

Purification and characterization of homogeneous assimilatory reduced nicotinamide adenine dinucleotide phosphate-nitrate reductase from Neurospora crassa.

Neurospora crassa wild type STA4 NADPH-nitrate reductase (NADPH : nitrate oxidoreductase, EC 1.6.6.3) has been purified 5000-fold with an overall yield of 25--50%. The final purified enzyme contained 4 associated enzymatic activities: NADPH-nitrate reductase, FADH2-nitrate reductase, reduced methyl viologen-nitrate reductase and NADPH-cytochrome c reductase. Polyacrylamide gel electrophoresis yielded 1 major and 1 minor protein band and both bands exhibited NADPH-nitrate and reduced methyl viologen-nitrate reductase activities. SDS gel electrophoresis yielded 2 protein bands corresponding to molecular weights of 115 000 and 130 000. A single N-terminal amino acid (glutamic acid) was found and proteolytic mapping for the two separated subunits appeared similar. Purified NADPH-nitrate reductase contained 1 mol of molybdenum and 2 mol of cytochrome b557 per mol protein. Non-heme iron, zinc and copper were not detectable. It is proposed that the Neurospora assimilatory NADPH-nitrate reductase consists of 2 similar cytochrome b557-containing 4.5-S subunits linked together by one molybdenum cofactor. A revised electron flow scheme is presented. p-Hydroxymercuribenzoate inhibition was reversed by sulfhydryl reagents. Inhibitory pattern of p-hydroxymercuribenzoate and phenylglyoxal revealed accessible sulfhydryl and arginyl residue(s) as functional group(s) in the earlier part of electron transport chain as possibly the binding site of NADPH or FAD.     

Further investigations regarding the role of trimethyllysine for cytochrome c uptake into mitochondria.

1. A mutant of the iso-1-cytochrome c gene from Saccharomyces cerevisiae has been constructed which contains an Arg codon, replacing the normal trimethylated Lys at position 77. 2. This mutated gene was cloned into a pGem 1 vector and used for the in vitro translation of yeast iso-1-cytochrome c. 3. Utilizing an in vitro mitochondria binding assay, it was found that the mutant cytochrome c could transverse the yeast mitochondrial membrane, however the amount of protein incorporated was 3-fold less that of the trimethylated wild type. 4. Omission of the protein methyltransferase from assays containing the wild type cytochrome c caused only a slight reduction (15%) in the amount of protein incorporated. 5. These results suggest while the lysine residue 77 of apocytochrome c is important for mitochondria uptake, the methylation of this residue seems to play a relatively minor role.     

Guanine nucleotide-dependent carboxyl methylation of mammalian membrane proteins

A guanine nucleotide-dependent protein carboxyl methylation is demonstrated in mammalian cell membranes. The methylation of membrane proteins of Mr 20,000-23,000 requires S-adenosylmethionine, GTP or nonhydrolyzable GTP analogs, and a cytoplasmic methyltransferase. The protein methyl groups are stable at neutral pH and under basic conditions hydrolyze to produce methanol. The specific methyl acceptor proteins and methyltransferases varied between tissues and cell types, suggesting that these methylations have cell-specific functions. The guanine nucleotide-dependent carboxyl methylations provide a possible mechanism for regulating the function of GTP-binding membrane proteins in the transduction of receptor-mediated signals of eukaryotic cells.     

Kinetic studies on redox reactions of hemoproteins. II. Reduction of thermoresistant cytochrome c-552 and horse heart cytochrome c by ascorbic acid.

The reductions of thermoresistant cytochrome c-552 and horse heart cytochrome c by ascorbic acid were studied by the stopped-flow method between pH 4 and 10. The results were as follows (1) The reduction of horse heart cytochrome c showed two relaxation decays above pH 8.5, one of which was pseudo-first order, as was the case below pH 8, while the other was nearly concentration-independent. These results were consistent with those reported by Greenwood and Palmer (J. Biol. Chem. (1965) 240, 3660-3663). (2) For the reduction of cytochrome c-552, only a single relaxational decay that obeyed pseudo-first order kinetics was observed. (3) It seems most reasonable to assume that the concentration-independent relaxation process can be attributed to the isomerization reaction accompanying ligand exchange, since it is known that only horse heart cytochrome c exhibits ligand exchange, involving a residue with pK 9.3.     

Partial purification and characterization of a protein lysine methyltransferase from plasmodia of Physarum polycephalum.

Plasmodia of Physarum polycephalum have an active protein lysine methyltransferase (S-adenosylmethionine:protein-lysine methyltransferase, EC 2.1.1.43). This enzyme has been purified 40-fold with a 13% yield, and it catalyzes the transfer of methyl groups from S-adenosyl-L-methionine to the epsilon-amino group of lysine residues with formation of N epsilon-mono-, N epsilon-di-, and N epsilon-trimethyllysines in a molar ratio of 4:1:1 based on [14C]methyl incorporation into the methylated lysines. The ratio remains unchanged at all stages of the partial purification, as well as after fractionation by sucrose density gradient centrifugation and gel electrophoresis. The rate of protein methylation is time dependent, enzyme concentration dependent, and requires the presence of a sulfhydryl reducing agent for optimal activity. The enzyme has optimal activity at pH 8 and is inhibited by S-adenosyl-L-homocysteine and EDTA. Lysine-rich and arginine-rich histones serve as the most effective exogenous protein acceptors; P. polycephalum actomyosin is inactive, and chick skeletal myofibrillar proteins are 25% as effective as exogenous mixed histones as substrates. Lysine, polylysine, ribonuclease A, cytochrome c, and bovine serum albumin are not methylated.     

Stoichiometric methylation of porcine adrenocorticotropin by protein carboxyl methyltransferase requires deamidation of asparagine 25. Evidence for methylation at the alpha-carboxyl group of atypical L-isoaspartyl residues

The enzymatic methylation of porcine adrenocorticotropin (ACTH) in both its native form and a form which is deamidated at asparagine 25 has been compared using purified protein carboxyl methyltransferase from bovine brain. Incubation of deamidated ACTH with high concentrations of methyltransferase resulted in near stoichiometric levels of methyl incorporation (78 mol %), while the methylation of native ACTH was highly substoichiometric (3-12 mol %). The Km and Vmax for deamidated ACTH were 1.9 microM and 11,200 pmol/min/mg, respectively, making this peptide the most specific substrate known for the mammalian methyltransferase. Deamidation of asparagine 25 leads to the formation of an atypical isopeptide bond in which the resulting aspartyl residue is linked to the adjacent glycine 26 via its side-chain beta-carboxyl group rather than the usual alpha-carboxyl linkage (Gráf, L., Bajusz, S., Patthy A., Barát, E., and Cseh, G. (1971) Acta Biochim. Biophys. Acad. Sci. Hung. 6, 415-418; Bornstein, P., and Balian, G. (1977) Methods Enzymol. 47, 132-145). A synthetic isopeptide (beta-linked) analog of deamidated ACTH serves as a highly effective substrate for the methyltransferase, but the corresponding normal (alpha-linked) peptide does not, indicating that this enzyme selectively recognizes the alpha-carboxyl group of atypical beta-linked L-aspartyl residues (see also accompanying paper (Murray, E.D., Jr., and Clarke, S. (1984) J. Biol. Chem. 259, 10722-10732]. Methylation of atypical beta-linked L-aspartyl residues resulting from deamidation can account for previous observations that in vitro protein carboxyl methylation in mammalian systems almost always occurs with a low stoichiometry and that these protein methyl esters are considerably less stable than most chemically formed protein methyl esters.     

Evidence for the amino-acid sequence of Crithidia fasciculata Cytochrome c555.

Evidence is presented which indicates that the amino acid sequence of cytochrome c555 from Crithidia fasciculata differs at sixteen positions from that of cytochrome c557 from Crithidia oncopelti. 101 residues were identified by dansyl-Edman degradation, carboxypeptidase digestion or considerations of the specificity of trypsin and of these, thirteen were found to be different from C. oncopelti cytochrome c557. The remaining 11 residues found in the amino acid composition of the trypic peptides were aligned on the basis of homology with cytochrome c557 and three further differences are proposed. The total of sixteen amino acid differences is surprising in view of the morphological and biochemical similarities of these organisms, and illustrates the problem of taxonomy of morphologically simple organisms. In both cytochromes only one cysteine residue is involved in the attachment of the protein to the prosthetic group.