TRNA methyltransferase

S-adenosylmethionine tRNA methyltransferases have been shown to occur in all kind of cells so far studied, both prokaryote and eukaryote. In eukaryote cells they are mostly localized in the cytosol and mitochondria recent results show the presence of these enzymes in nuclei of mouse L-cells and of Xenopus oocyte l. These latter results suggest that the intracellular localization of such enzymes should be more accurately investigated by the refined techniques now available. The possible localization of these enzymes within the nuclei is of interest if visualized in connection with the timing and the place of tRNA maturation events. 

Many difficulties have to be faced during the purification of tRNA methyltransferases. The major ones are ii) enzyme multiplicity, which makes it cumbersome to isolate each specific reaction product, and then difficult to evaluate specific activity and extent of purification (ii) enzyme instability (Hi) frequent contamination by ribonuclease activity (iv) possible presence of complexes with endogenous tRNA (v) unavailability of a proper tRNA substrate, as we have already discussed. To point out the problem of... 

The molecular size of the four methylase activities isolated from S.typhimurium was studied by comparing their sedimentation profile with that of proteins of known molecular weight. Results obtained show that four tRNA methylases have a Mj- ranging from 25,000 to 65,000 daltons, except the enzyme preparation mentioned above as no. 1, which shows also an aggregate form of higher (120,000). All the other tRNA methyltransferases isolated so far from other sources appear to have higher. ... 

Adenosylhomocysteine (Ado-Hcy) is the first studied and the most active inhibitor of tRNA methyltransferases, both from... 

METHYLENETETRAHYDROFOLATE tRNA (URACIL-5-)-METHYLTRANSFERASE (FADH2 OXIDIZING)... 

Methyltransferases that utilize S-adenosyl-L-methionine as the methyl donor (and thus generating S-adenosyl-L-homocysteine) catalyze (a) A-methylation (e.g., norepinephrine methyltransferase, histamine methyltransferase, glycine methyltransferase, and DNA-(adenine-A ) methyltransferase), (b) O-methylation (e.g., acetylsero-tonin methyltransferase, catechol methyltransferase, and tRNA-(guanosine-0 ) methyltransferase), (c) S-methyl-ation (e.g., thiopurine methyltransferase and methionine S-methyltransferase), (d) C-methylation (eg., DNA-(cy-tosine-5) methyltransferase and indolepyruvate methyltransferase), and even (e) Co(II)-methylation during the course of the reaction catalyzed by methionine syn-thase. ... 

Santi DV, Hardy LW. Catalytic mechanism and inhibition of tRNA (uracil-5-)methyltransferase evidence for covalent catalysis. Biochemistry 1987 26 8599-8606. 

Comprehensive Biological Catalysis—a Mechanistic Reference Volume has recently been published. The fiiU contents list (approximate number of references in parentheses) is as follows S-adenosylmethionine-dependent methyltransferases (110) prenyl transfer and the enzymes of terpenoid and steroid biosynthesis (330) glycosyl transfer (800) mechanism of folate-requiring enzymes in one-carbon metabohsm (260) hydride and alkyl group shifts in the reactions of aldehydes and ketones (150) phosphoenolpyruvate as an electrophile carboxyvinyl transfer reactions (140) physical organic chemistry of acyl transfer reactions (220) catalytic mechanisms of the aspartic proteinases (90) the serine proteinases (135) cysteine proteinases (350) zinc proteinases (200) esterases and lipases (160) reactions of carbon at the carbon dioxide level of oxidation (390) transfer of the POj group (230) phosphate diesterases and triesterases (160) ribozymes (70) catalysis of tRNA aminoacylation by class I and class II aminoacyl-tRNA synthetases (220) thio-disulfide exchange of divalent sulfirr (150) and sulfotransferases (50). 

A similar chemo-enzymatic approach has been used for fluorescent labeling of tRNA in vitro [84]. The tRNA-specific methyltransferase Trml was employed to transfer a pent-2-en-4-ynyl group from a modified AdoMet analogue to the N position of guanosine 26 in tRNA for subsequent CuAAC labeling [84]. 

A second group of SAM-dependent methylases acts on much larger substrates, such as proteins and nucleic acids (9). Only recently has progress been made on isolating purified proteins which methylate macromolecules such as tRNA (10), mRNA (11), and proteins (12). In the case of tRNA (adenine-1)-methyltransferase (E.C. 2.1.1.36) (10a) and protein carboxyl-0-methyltransferase (E.C. 2.1.1.24) (13), kinetic studies are consistent with the random sequential reaction proposed for COMT, and thus suggest a direct methyl transfer in the ternary complex. One enzyme which does not show kinetics consistent with a direct methyl transfer is histamine-N-rnethyltransferase (E. C. 2.1.1.8) (14). The data reported are consistent with a double—displacement mechanism of... 

The Michaelis constants for adenosylmethionine of tRNA methylases vary from 7 X 10 M for E.ooli tRNA(guanine-1)-methyltransferase to 1.5 X 10 M for mammalian enzymes , xhe four S,typhinrurium enzymes separated in the authors laboratory s show values ranging from 1.5 to 3.2 x 10 M. While the apparent affinity constants for adenosylmethionine are very similar, those for undermethylated tRNA appear to be scattered in a wider range for example, the K] s found for the four enzymes isolated from S. typhirnurivm range from 1.2 x 10" M to 6.3 x 10 M. This could imply that the site for adenosylmethionine is similar in all the different enzymes, whereas interaction with tRNA involves the presence of enzyme sites, which are more typical for specific methylatable sites of tRNA. It must be noted, however, that the measure of for tRNA can be strongly influenced by the use of a non suitable substrate, like crude undermethylated tRNA, in which it is difficult to assess precisely what proportion of the entire preparation is the true substrate. 

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