A Novel 3-Methylhistidine Modification of Yeast Ribosomal Protein Rpl3 Is Dependent upon the YIL110W Methyltransferase

We have shown that Rpl3, a protein of the large ribosomal subunit from baker''s yeast (Saccharomyces cerevisiae), is stoichiometrically monomethylated at position 243, producing a 3-methylhistidine residue. This conclusion is supported by top-down and bottom-up mass spectrometry of Rpl3, as well as by biochemical analysis of Rpl3 radiolabeled in vivo with S-adenosyl-l-[methyl-³H]methionine. The results show that a +14-Da modification occurs within the GTKKLPRKTHRGLRKVAC sequence of Rpl3. Using high-resolution cation-exchange chromatography and thin layer chromatography, we demonstrate that neither lysine nor arginine residues are methylated and that a 3-methylhistidine residue is present. Analysis of 37 deletion strains of known and putative methyltransferases revealed that only the deletion of the YIL110W gene, encoding a seven β-strand methyltransferase, results in the loss of the +14-Da modification of Rpl3. We suggest that YIL110W encodes a protein histidine methyltransferase responsible for the modification of Rpl3 and potentially other yeast proteins, and now designate it Hpm1 (Histidine protein methyltransferase 1). Deletion of the YIL110W/HPM1 gene results in numerous phenotypes including some that may result from abnormal interactions between Rpl3 and the 25 S ribosomal RNA. This is the first report of a methylated histidine residue in yeast cells, and the first example of a gene required for protein histidine methylation in nature.     

Methylation of translation-associated proteins in Saccharomyces cerevisiae: Identification of methylated lysines and their methyltransferases

This study aimed to identify sites of lysine methylation in Saccharomyces cerevisiae and the associated methyltransferases. Hexapeptide ligand affinity chromatography was used to normalize the abundance levels of proteins in whole cell lysate. MS/MS, in association with antibody-based detection, was then used to identify lysine methylated proteins and the precise sites of modification. Lysine methylation was found on the proteins elongation factor (EF) 1-α, 2, and 3A, as well as ribosomal proteins 40S S18-A/B, 60S L11-A/B, L18-A/B, and L42-A/B. Precise sites were mapped in all cases. Single-gene knockouts of known and putative methyltransferase(s), in association with MS/MS, showed that EF1-α is monomethylated by Efm1 at lysin 30 and dimethylated by See1 at lysine 316. Methyltransferase Rkm1 was found to monomethylate 40S ribosomal protein S18-A/B at lysine 48. Knockout analysis also revealed that putative methyltransferase YBR271W affects the methylation of proteins EF2 and 3A; this was detected by Western blotting and immunodetection. This methyltransferase shows strong interspecies conservation and a tryptophan-containing motif associated with its active site. We suggest that enzyme YBR271W is named EF methyltransferase 2 (Efm2), in line with the recent naming of YHL039W as Efm1.     

Novel N-terminal and Lysine Methyltransferases That Target Translation Elongation Factor 1A in Yeast and Human

Eukaryotic elongation factor 1A (eEF1A) is an essential, highly methylated protein that facilitates translational elongation by delivering aminoacyl-tRNAs to ribosomes. Here, we report a new eukaryotic protein N-terminal methyltransferase, Saccharomyces cerevisiae YLR285W, which methylates eEF1A at a previously undescribed high-stoichiometry N-terminal site and the adjacent lysine. Deletion of YLR285W resulted in the loss of N-terminal and lysine methylation in vivo, whereas overexpression of YLR285W resulted in an increase of methylation at these sites. This was confirmed by in vitro methylation of eEF1A by recombinant YLR285W. Accordingly, we name YLR285W as elongation factor methyltransferase 7 (Efm7). This enzyme is a new type of eukaryotic N-terminal methyltransferase as, unlike the three other known eukaryotic N-terminal methyltransferases, its substrate does not have an N-terminal [A/P/S]-P-K motif. We show that the N-terminal methylation of eEF1A is also present in human; this conservation over a large evolutionary distance suggests it to be of functional importance. This study also reports that the trimethylation of Lys⁷⁹ in eEF1A is conserved from yeast to human. The methyltransferase responsible for Lys⁷⁹ methylation of human eEF1A is shown to be N6AMT2, previously documented as a putative N(6)-adenine-specific DNA methyltransferase. It is the direct ortholog of the recently described yeast Efm5, and we show that Efm5 and N6AMT2 can methylate eEF1A from either species in vitro. We therefore rename N6AMT2 as eEF1A-KMT1. Including the present work, yeast eEF1A is now documented to be methylated by five different methyltransferases, making it one of the few eukaryotic proteins to be extensively methylated by independent enzymes. This implies more extensive regulation of eEF1A by this posttranslational modification than previously appreciated.Protein methylation is emerging as one of the most prominent posttranslational modifications in the eukaryotic cell (1). Often showing high evolutionary conservation, it is increasingly recognized for its role in modulating protein–protein interactions (2). Indeed, it has been documented in protein interaction codes (3), such as those of the histones and p53 (4, 5), where it shows interplay with modifications such as acetylation and phosphorylation. Despite this, there remains a paucity of understanding of the enzymes that catalyze protein methylation. Many of the known methyltransferases target histones. However, many other methyltransferases have been discovered recently that act on nonhistone proteins (6).While protein methylation predominantly occurs on lysine and arginine residues, it is also known to occur on glutamine, asparagine, glutamate, histidine, cysteine, and the N- and C termini of proteins. Although the presence of N-terminal methylation on numerous proteins has been known for decades (7), the first enzymes responsible for this methylation have only recently been discovered (8, 9). The Saccharomyces cerevisiae protein Tae1 and its human ortholog N-terminal methyltransferase 1 (NTMT1) catalyze N-terminal methylation of proteins with an N-terminal [A/P/S]-P-K motif (after methionine removal). Yet there is evidence that these enzymes may recognize a more general N-terminal motif (10). Human NTMT2 is a monomethyltransferase that methylates the same substrates as NTMT1 and may prime substrate proteins with monomethylation to assist subsequent trimethylation by NTMT1 (11).The biological function of N-terminal methylation on some proteins has been recently revealed. For example, N-terminal methylation of regulator of chromatin condensation protein 1 (RCC1) is known to affect its binding to chromatin and thereby the correct chromosomal segregation during mitosis (12, 13), and N-terminal methylation of DNA damage-binding protein 2 (DDB2) is important for its role in UV-damaged DNA repair (14). Interestingly, there is evidence of interplay between N-terminal methylation and other posttranslational modifications (15), suggesting that, like lysine and arginine methylation, it may be incorporated into protein interaction codes (3). N-terminal methylation therefore appears to be a modification of functional importance in the cell.Eukaryotic elongation factor 1A (eEF1A), and its bacterial ortholog EF-Tu, is an essential translation elongation factor that is found in all living organisms. Its canonical function is in facilitating delivery of aminoacyl-tRNAs to the ribosome; however, it is also known to have a role in many other cellular functions, such as actin bundling, nuclear export, and proteasomal degradation (16). A number of methyltransferases have been discovered in both S. cerevisiae and human that target translation elongation factors. In yeast, four of these elongation factor methyltransferases (EFMs) act on eEF1A, namely Efm1, Efm4, Efm5, and Efm6, generating monomethylated Lys³⁰, dimethylated Lys³¹⁶, trimethylated Lys⁷⁹, and monomethylated Lys³⁹⁰, respectively (17–19). Human METTL10 is the ortholog of Efm4 in that it trimethylates eEF1A at Lys³¹⁸, which is equivalent to Lys³¹⁶ in yeast (20). Interestingly, eukaryotic elongation factor 2 (eEF2) is also methylated by a number of lysine methyltransferases. In yeast, Efm2 and Efm3 act on eEF2, generating dimethylated Lys⁶¹³ and trimethylated Lys⁵⁰⁹, respectively (21–24). Human eEF2-KMT is the ortholog of Efm3 in that it trimethylates eEF2 at Lys⁵²⁵, which is equivalent to Lys⁵⁰⁹ in yeast eEF2 (23).Here, we report the N-terminal methylation of eEF1A in S. cerevisiae and the identification of the methyltransferase that catalyzes this event. Using parallel reaction monitoring and MS/MS/MS (MS3), we unambiguously localize the modification to the N-terminal glycine and show it is conserved in the human cell. We also show that YLR285W, which we rename elongation factor methyltransferase 7 (Efm7), is responsible for this modification in yeast, as well as dimethylation at the adjacent lysine. We also characterize the methyltransferases responsible for methylation of lysine 79 in eEF1A. Human N6AMT2 is shown to be the ortholog of yeast Efm5 through its capacity to methylate yeast and human eEF1A at Lys⁷⁹ in vitro. We therefore rename N6AMT2 as eEF1A-KMT1.     

Elongation factor methyltransferase 3 – A novel eukaryotic lysine methyltransferase

Here we describe the discovery of Saccharomycescerevisiae protein YJR129Cp as a new eukaryotic seven-beta-strand lysine methyltransferase. An immunoblotting screen of 21 putative methyltransferases showed a loss in the methylation of elongation factor 2 (EF2) on knockout of YJR129C. Mass spectrometric analysis of EF2 tryptic peptides localised this loss of methylation to lysine 509, in peptide LVEGLKR. In vitro methylation, using recombinant methyltransferases and purified EF2, validated YJR129Cp as responsible for methylation of lysine 509 and Efm2p as responsible for methylation at lysine 613. Contextualised on previously described protein structures, both sites of methylation were found at the interaction interface between EF2 and the 40S ribosomal subunit. In line with the recently discovered Efm1 and Efm2 we propose that YJR129C be named elongation factor methyltransferase 3 (Efm3). The human homolog of Efm3 is likely to be the putative methyltransferase FAM86A, according to sequence homology and multiple lines of literature evidence.     

The ribosomal l1 protuberance in yeast is methylated on a lysine residue catalyzed by a seven-beta-strand methyltransferase

Modification of proteins of the translational apparatus is common in many organisms. In the yeast Saccharomyces cerevisiae, we provide evidence for the methylation of Rpl1ab, a well conserved protein forming the ribosomal L1 protuberance of the large subunit that functions in the release of tRNA from the exit site. We show that the intact mass of Rpl1ab is 14 Da larger than its calculated mass with the previously described loss of the initiator methionine residue and N-terminal acetylation. We determined that the increase in mass of yeast Rpl1ab is consistent with the addition of a methyl group to lysine 46 using top-down mass spectrometry. Lysine modification was confirmed by detecting (3)H-N-ε-monomethyllysine in hydrolysates of Rpl1ab purified from yeast cells radiolabeled in vivo with S-adenosyl-l-[methyl-(3)H]methionine. Mass spectrometric analysis of intact Rpl1ab purified from 37 deletion strains of known and putative yeast methyltransferases revealed that only the deletion of the YLR137W gene, encoding a seven-β-strand methyltransferase, results in the loss of the +14-Da modification. We expressed the YLR137W gene as a His-tagged protein in Escherichia coli and showed that it catalyzes N-ε-monomethyllysine formation within Rpl1ab on ribosomes from the ΔYLR137W mutant strain lacking the methyltransferase activity but not from wild-type ribosomes. We also showed that the His-tagged protein could catalyze monomethyllysine formation on a 16-residue peptide corresponding to residues 38-53 of Rpl1ab. We propose that the YLR137W gene be given the standard name RKM5 (ribosomal lysine (K) methyltransferase 5). Orthologs of RKM5 are found only in fungal species, suggesting a role unique to their survival.     

On your histone mark,SET, methylate!

Lysine methylation of histones and non-histone proteins has emerged in recent years as a posttranslational modification with wide-ranging cellular implications beyond epigenetic regulation. The molecular interactions between lysine methyltransferases and their substrates appear to be regulated by posttranslational modifications surrounding the lysine methyl acceptor. Two very interesting examples of this cross-talk between methyl-lysine sites are found in the SET (Su(var)3–9, Enhancer-of-zeste, Trithorax) domain-containing lysine methyltransferases SET7 and SETDB1, whereby the histone H3 trimethylated on lysine 4 (H3K4^me3) modification prevents methylation by SETDB1 on H3 lysine 9 (H3K9) and the histone H3 trimethylated on lysine 9 (H3K9^me3) modification prevents methylation by SET7 on H3K4. A similar cross-talk between posttranslational modifications regulates the functions of non-histone proteins such as the tumor suppressor p53 and the DNA methyltransferase DNMT1. Herein, in cis effects of acetylation, phosphorylation, as well as arginine and lysine methylation on lysine methylation events will be discussed.     

Yeast ribosomal/cytochrome c SET domain methyltransferase subfamily: identification of Rpl23ab methylation sites and recognition motifs

Ribosomal protein L23ab is specifically dimethylated at two distinct sites by the SET domain-containing enzyme Rkm1 in the yeast Saccharomyces cerevisiae. Using liquid column chromatography with electrospray-ionization mass spectrometry, we determined that Rpl23ab purified from the Deltarkm1 deletion strain demonstrated a loss in mass of approximately 56 Da when compared with Rpl23ab purified from the wild type strain. When Rpl23ab was proteolyzed, using proteinase ArgC or CNBr, and the peptides derived were analyzed by tandem mass spectrometry, no sites of methylation were found in Rpl23ab purified from the Deltarkm1 deletion strain, whereas two sites of dimethylation were observed in the wild type strain at lysine residues 105 and 109. We show that both Rpl23a and Rpl23b are expressed and methylated in vivo in yeast. Using polysomal fractionation, we demonstrate that the deletion of RKM1 has no effect on ribosomal complex formation. Comparison of SET domain methyltransferase substrates in yeast reveal sequence similarities around the lysine methylation sites and suggest that an (Asn/Pro)-Pro-Lys consensus sequence may be a target for methylation by subfamily 2 SET domain methyltransferases. Finally, we show the presence of Rkm1 homologs in fungi, plants, and mammals including humans.     

The active site of the SET domain is constructed on a knot

The SET domain contains the catalytic center of lysine methyltransferases that target the N-terminal tails of histones and regulate chromatin function. Here we report the structure of the SET7/9 protein in the absence and presence of its cofactor product, S-adenosyl-L-homocysteine (AdoHcy). A knot within the SET domain helps form the methyltransferase active site, where AdoHcy binds and lysine methylation is likely to occur. A structure-guided comparison of sequences within the SET protein family suggests that the knot substructure and active site environment are conserved features of the SET domain.     

A novel SET domain methyltransferase in yeast: Rkm2-dependent trimethylation of ribosomal protein L12ab at lysine 10

The ribosomal protein L12ab (Rpl12ab) in Saccharomyces cerevisiae is modified by methylation at both arginine and lysine residues. Although the enzyme responsible for the modification reaction at arginine 66 has been identified (Rmt2), the enzyme(s) responsible for the lysine modification(s) has not been found, and the site(s) of methylation has not been determined. Here we demonstrate, using a combination of mass spectrometry and labeling assays, that the yeast gene YDR198c encodes the enzyme responsible for the predominant epsilon-trimethylation at lysine 10 in Rpl12ab. An additional site of predominant epsilon-dimethylation is observed at lysine 3; the enzyme catalyzing this modification is not known. The YDR198c gene encodes a SET domain similar to that of the Rkm1 enzyme responsible for modifying Rpl23ab, and we have now designated the YDR198c gene product as Rkm2 (ribosomal lysine methyltransferase 2). The effect of the loss of the enzyme on ribosomal complex stability was studied by polysomal fractionation. However, no difference was observed between the Deltarkm2 deletion strain and its parent wild type strain. With the identification of this enzyme, it appears that the 12 SET domain family members in yeast can now be divided into two subfamilies based on function and amino acid sequence identity. One branch includes enzymes that modify histones, including Set1 and Set2; the other branch includes Rkm1, Rkm2, and Ctm1, the cytochrome c methyltransferase. These studies suggest that the remaining seven SET domain proteins may also be lysine methyltransferases.     

WRAD: enabler of the SET1-family of H3K4 methyltransferases

Methylation of histone H3 at lysine 4 (H3K4) is a conserved feature of active chromatin catalyzed by methyltransferases of the SET1-family (SET1A, SET1B, MLL1, MLL2, MLL3 and MLL4 in humans). These enzymes participate in diverse gene regulatory networks with a multitude of known biological functions, including direct involvement in several human disease states. Unlike most lysine methyltransferases, SET1-family enzymes are only fully active in the context of a multi-subunit complex, which includes a protein module comprised of WDR5, RbBP5, ASH2L and DPY-30 (WRAD). These proteins bind in close proximity to the catalytic SET domain of SET1-family enzymes and stimulate H3K4 methyltransferase activity. The mechanism by which WRAD promotes catalysis involves elements of allosteric control and possibly the utilization of a second H3K4 methyltransferase active site present within WRAD itself. WRAD components also engage in physical interactions that recruit SET1-family proteins to target sites on chromatin. Here, the known molecular mechanisms through which WRAD enables the function of SET1-related enzymes will be reviewed.     

Toward the development of potent and selective bisubstrate inhibitors of protein arginine methyltransferases

Prototype inhibitors of protein arginine methyltransferases (PRMTs) have been constructed by attaching guanidine functionality via a variable linker to non-reactive amine analogues of the cellular co-factor (S)-adenosyl methionine (AdoMet). Potent inhibition of PRMT1 (IC₅₀ of ～3–6 μM) combined with weak inhibition of the lysine methyltransferase SET7 (～50% of activity at 100 μM) was observed for two such compounds.     

Two Protein Lysine Methyltransferases Methylate Outer Membrane Protein B from Rickettsia

Rickettsia prowazekii, the etiologic agent of epidemic typhus, is a potential biological threat agent. Its outer membrane protein B (OmpB) is an immunodominant antigen and plays roles as protective envelope and as adhesins. The observation of the correlation between methylation of lysine residues in rickettsial OmpB and bacterial virulence has suggested the importance of an enzymatic system for the methylation of OmpB. However, no rickettsial lysine methyltransferase has been characterized. Bioinformatic analysis of genomic DNA sequences of Rickettsia identified putative lysine methyltransferases. The genes of the potential methyltransferases were synthesized, cloned, and expressed in Escherichia coli, and expressed proteins were purified by nickel-nitrilotriacetic acid (Ni-NTA) affinity chromatography. The methyltransferase activities of the purified proteins were analyzed by methyl incorporation of radioactively labeled S-adenosylmethionine into recombinant fragments of OmpB. Two putative recombinant methyltransferases (rRP789 and rRP027-028) methylated recombinant OmpB fragments. The specific activity of rRP789 is 10- to 30-fold higher than that of rRP027-028. Western blot analysis using specific antibodies against trimethyl lysine showed that both rRP789 and rRP027-028 catalyzed trimethylation of recombinant OmpB fragments. Liquid chromatography-tandem mass spectrometry (LC/MS-MS) analysis showed that rRP789 catalyzed mono-, di-, and trimethylation of lysine, while rRP027-028 catalyzed exclusively trimethylation. To our knowledge, rRP789 and rRP027-028 are the first biochemically characterized lysine methyltransferases of outer membrane proteins from Gram-negative bacteria. The production and characterization of rickettsial lysine methyltransferases provide new tools to investigate the mechanism of methylation of OmpB, effects of methylation on the structure and function of OmpB, and development of methylated OmpB-based diagnostic assays and vaccine candidates.     

Characterization of the PR domain of RIZ1 histone methyltransferase

RIZ1 (PRDM2) and PRDI-BF1 (PRDM1) are involved in B cell differentiation and the development of B cell lymphomas. These proteins are expressed in two forms that differ by the presence or absence of a PR domain. The protein product that retains the PR domain is anti-tumorigenic while the product that lacks the PR domain is oncogenic and over-expressed in tumor cells. The conserved PR domain is homologous to the SET domain from a family of histone methyltransferases. RIZ1 is also a histone methyltransferase and methylates lysine 9 in histone H3. This activity has been mapped to the PR domain. In the present study, deuterium exchange mass spectrometry was used to define the structural boundaries of the RIZ1 PR domain and to map sites of missense mutations that occur in human cancers and reduce methyltransferase activity. Flexible segments were selectively deleted to produce protein products that crystallize for structural studies. Segments at the carboxyl terminus of the PR domain that are involved in methylation of H3 were shown to be flexible, similar to SET domains, suggesting that the PR and SET methyltransferases may belong to an emerging class of proteins that contain mobile functional regions.     

Saccharomyces cerevisiae Eukaryotic Elongation Factor 1A (eEF1A) Is Methylated at Lys-390 by a METTL21-Like Methyltransferase

The human methyltransferases (MTases) METTL21A and VCP-KMT (METTL21D) were recently shown to methylate single lysine residues in Hsp70 proteins and in VCP, respectively. The yet uncharacterized MTase encoded by the YNL024C gene in Saccharomyces cerevisiae shows high sequence similarity to METTL21A and VCP-KMT, as well as to their uncharacterized paralogues METTL21B and METTL21C. Despite being most similar to METTL21A, the Ynl024c protein does not methylate yeast Hsp70 proteins, which were found to be unmethylated on the relevant lysine residue. Eukaryotic translation elongation factor eEF1A in yeast has been reported to contain four methylated lysine residues (Lys30, Lys79, Lys318 and Lys390), and we here show that the YNL024C gene is required for methylation of eEF1A at Lys390, the only of these methylations for which the responsible MTase has not yet been identified. Lys390 was found in a partially monomethylated state in wild-type yeast cells but was exclusively unmethylated in a ynl024cΔ strain, and over-expression of Ynl024c caused a dramatic increase in Lys390 methylation, with trimethylation becoming the predominant state. Our results demonstrate that Ynl024c is the enzyme responsible for methylation of eEF1A at Lys390, and in accordance with prior naming of similar enzymes, we suggest that Ynl024c is renamed to Efm6 (Elongation factor MTase 6).     

Identification and Characterization of a Novel Evolutionarily Conserved Lysine-specific Methyltransferase Targeting Eukaryotic Translation Elongation Factor 2 (eEF2)

The components of the cellular protein translation machinery, such as ribosomal proteins and translation factors, are subject to numerous post-translational modifications. In particular, this group of proteins is frequently methylated. However, for the majority of these methylations, the responsible methyltransferases (MTases) remain unknown. The human FAM86A (family with sequence similarity 86) protein belongs to a recently identified family of protein MTases, and we here show that FAM86A catalyzes the trimethylation of eukaryotic elongation factor 2 (eEF2) on Lys-525. Moreover, we demonstrate that the Saccharomyces cerevisiae MTase Yjr129c, which displays sequence homology to FAM86A, is a functional FAM86A orthologue, modifying the corresponding residue (Lys-509) in yeast eEF2, both in vitro and in vivo. Finally, Yjr129c-deficient yeast cells displayed phenotypes related to eEF2 function (i.e. increased frameshifting during protein translation and hypersensitivity toward the eEF2-specific drug sordarin). In summary, the present study establishes the function of the previously uncharacterized MTases FAM86A and Yjr129c, demonstrating that these enzymes introduce a functionally important lysine methylation in eEF2. Based on the previous naming of similar enzymes, we have redubbed FAM86A and Yjr129c as eEF2-KMT and Efm3, respectively.     

Selenium-Based S-Adenosylmethionine Analog Reveals the Mammalian Seven-Beta-Strand Methyltransferase METTL10 to Be an EF1A1 Lysine Methyltransferase

Lysine methylation has been extensively studied in histones, where it has been shown to provide specific epigenetic marks for the regulation of gene expression; however, the molecular mechanism and physiological function of lysine methylation in proteins other than histones remains to be fully addressed. To better understand the substrate diversity of lysine methylation, S-adenosylmethionine (SAM) derivatives with alkyne-moieties have been synthesized. A selenium-based SAM analog, propargylic Se-adenosyl-l-selenomethionine (ProSeAM), has a wide spectrum of reactivity against various lysine methyltransferases (KMTs) with sufficient stability to support enzymatic reactions in vitro. By using ProSeAM as a chemical probe for lysine methylation, we identified substrates for two seven-beta-strand KMTs, METTL21A and METTL10, on a proteomic scale in mammalian cells. METTL21A has been characterized as a heat shock protein (HSP)-70 methyltransferase. Mammalian METTL10 remains functionally uncharacterized, although its ortholog in yeast, See1, has been shown to methylate the translation elongation factor eEF1A. By using ProSeAM-mediated alkylation followed by purification and quantitative MS analysis, we confirmed that METTL21A labels HSP70 family proteins. Furthermore, we demonstrated that METTL10 also methylates the eukaryotic elongation factor EF1A1 in mammalian cells. Subsequent biochemical characterization revealed that METTL10 specifically trimethylates EF1A1 at lysine 318 and that siRNA-mediated knockdown of METTL10 decreases EF1A1 methylation levels in vivo. Thus, our study emphasizes the utility of the synthetic cofactor ProSeAM as a chemical probe for the identification of non-histone substrates of KMTs.     

Kinetic manifestation of processivity during multiple methylations catalyzed by SET domain protein methyltransferases

Processive versus distributive methyl group transfer was assessed for pea Rubisco large subunit methyltransferase, a SET domain protein lysine methyltransferase catalyzing the formation of trimethyllysine-14 in the large subunit of Rubisco. Catalytically competent complexes between an immobilized form of des(methyl) Rubisco and Rubisco large subunit methyltransferase were used to demonstrate enzyme release that was co-incident with and dependent on formation of trimethyllysine. Catalytic rate constants determined for formation of trimethyllysine were considerably lower ( approximately 10-fold) than rate constants determined for total radiolabel incorporation from [3H-methyl]-S-adenosylmethionine. Double-reciprocal velocity plots under catalytic conditions favoring monomethyllysine indicated a random or ordered reaction mechanism, while conditions favoring trimethyllysine suggested a hybrid ping-pong mechanism. These results were compared with double-reciprocal velocity plots and product analyses obtained for HsSET7/9 (a monomethyltransferase) and SpCLR4 (a dimethyltransferase) and suggest a predictive ability of double-reciprocal velocity plots for single versus multiple methyl group transfers by SET domain protein lysine methyltransferases. A model is proposed for SET domain protein lysine methyltransferases in which initial binding of polypeptide substrate and S-adenosylmethionine is random, with polypeptide binding followed by deprotonation of the epsilon-amine of the target lysyl residue and subsequent methylation. Following methyl group transfer, S-adenosylhomocysteine and monomethylated polypeptide dissociate from monomethyltransferases, but di- and trimethyltransferases begin a successive and catalytically obligatory deprotonation of enzyme-bound methylated lysyl intermediates, which along with binding and release of S-adenosylmethionine and S-adenosylhomocysteine is manifested as a hybrid ping-pong-like reaction mechanism.