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.
Dirk
Lynnette M A
LM
Department of Horticulture, Plant Physiology/Biochemistry/Molecular Biology Program, 401D Plant Science Building, University of Kentucky, Lexington, Kentucky 40546-0312, USA.
Flynn
E Megan
EM
Dietzel
Kevin
K
Couture
Jean-François
JF
Trievel
Raymond C
RC
Houtz
Robert L
RL
D000241:Adenosine / Q000031:analogs & derivatives / Q000737:chemistry; D000818:Animals; D002384:Catalysis; D002417:Cattle; D018797:Cell Cycle Proteins / Q000737:chemistry*; D000076983:Histone Methyltransferases; D011495:Histone-Lysine N-Methyltransferase / Q000737:chemistry*; D007700:Kinetics; D008239:Lysine / Q000031:analogs & derivatives / Q000737:chemistry; D008745:Methylation; D008780:Methyltransferases / Q000737:chemistry*; D008956:Models, Chemical; D009097:Multienzyme Complexes / Q000737:chemistry*; D018532:Pisum sativum / Q000201:enzymology*; D010940:Plant Proteins / Q000737:chemistry*; D011496:Protein Methyltransferases; D017434:Protein Structure, Tertiary; D012435:S-Adenosylhomocysteine; D029702:Schizosaccharomyces pombe Proteins / Q000737:chemistry*
D018797:Cell Cycle Proteins; D009097:Multienzyme Complexes; D010940:Plant Proteins; D029702:Schizosaccharomyces pombe Proteins; C003712:trimethyllysine; D012435:S-Adenosylhomocysteine; D000076983:Histone Methyltransferases; D008780:Methyltransferases; D011496:Protein Methyltransferases; C097344:ribulose-1,5-bisphosphate carboxylase-oxygenase large subunit epsilonN-methyltransferase; D011495:Histone-Lysine N-Methyltransferase; C112744:clr4 protein, S pombe; D008239:Lysine; D000241:Adenosine; C006235:sinefungin