The configuration at the asymmetric sulfonium pole of S-adenosyl-L-methionine (SAM) necessary for optimal enzymatic binding and methyl donation has been elucidated in this study. For the transmethylations catalyzed by catechol O-methyltransferase, phenylethanolamine N-methyltransferase, histamine N-methyltransferase, and hydroxyindole O-methyltransferase, it was demonstrated that only the natural (-) enantiomer of SAM was active as a methyl donor. The corresponding (+)-SAM, which was prepared by enzymatic resolution of synthetic (+/-)-SAM, was shown to be inactive as a methyl donor in these enzymatic reactions. The (+)-SAM was found, however, to be a potent inhibitor of each of these enzyme-catalyzed transmethylations. These results suggest that the (+) enantiomer offers a nonproductive configuration for the methyl-transfer reaction itself; however, this configuration fails to hamper enzymatic binding. These results are discussed relative to the geometric requirements necessary for the methyl-transfer reaction and the requirements for enzymatic binding.

Peptides and peptide-like molecules as a class have very poor permeability through biological membranes, which severely compromises their potential effectiveness as therapeutic agents. In order to gain insight into the problem of delivering peptide and protein drugs and to establish a model in which the effects of systematic structural variations on transport can be explored, an investigation of the solution conformation of a membrane-permeable peptide was undertaken. Delta-sleep-inducing peptide (DSIP, MW 849) was used in this investigation. DSIP is a charged, hydrophilic peptide that possesses the unusual ability to diffuse passively across the blood-brain barrier (BBB) in vivo [Kastin, A. J., Banks, W. A., Castellanos, P. F., Nissen, C., & Coy, D. H. (1982) Pharmacol. Biochem. Behav. 17, 1187-1191] and across monolayers of brain microvessel endothelial cells in vitro, a model of the BBB [Raeissi, S., & Audus, K. L. (1989) J. Pharm. Pharmacol. 41, 848-852]. This nonapeptide was studied in solution using one- and two-dimensional nuclear magnetic resonance (NMR), circular dichroism (CD), Fourier transform infrared (FT-IR), and fluorescence spectroscopies in conjunction with molecular modeling. Our spectroscopic findings suggest that DSIP exists in a dynamic equilibrium between unordered and folded structures. Residues 2-5 and 6-9 tend to form type I beta-turns in aqueous solution and a similar, but more ordered, helix-like structure inducible in 40% trifluoroethanol (TFE). NMR, FT-IR, and CD studies in aqueous solution support the dynamic equilibrium hypothesis with the IR data, suggesting that the beta-turn population is approximately 40%.(ABSTRACT TRUNCATED AT 250 WORDS)

Structural analogues of S-adenosyl-L-homocysteine (L-SAH), with modifications in the amino acid or base portions of the molecule, have been synthesized and evaluated for their abilities to inhibit the transmethylations catalyzed by catechol O-methyltransferase (COMT), phenylethanolamine N-methyltransferase (PNMT), histamine N-methyltransferase (HMT), hydroxyindole O-methyltransferase (HIOMT), and indoleethylamine N-methyltransferase (INMT). From these studies some interesting and potentially useful differences in the structural features of L-SAH needed to produce maximal binding to these methyltransferases were detected. This paper provides evidence that 8-azaadenosyl-L-homocysteine is a potent and selective inhibitor of HIOMT, whereas N6-methyladenosyl-L-homocysteine and N6-methyl-3-deazaadenosyl-L-homocysteine are selective inhibitors in INMT. In contrast, it was found that S-tubercidinyl-L-homocysteine was a fairly potent, but nonselective inhibitor of all of the methyltransferases studied. The differences and the similarities in the requirements for the binding of SAH to methyltransferases which were detected in this study and earlier studies from our laboratory, are described. The possibilites of utilizing differences in binding requirements for the design of SAH analogues as specific inhibitors of methyltransferases are discussed.

Structural analogues of S-adenosyl-L-methionine (SAM), with modifications in the amino acid, sugar, or base portions of the molecule, have been synthesized and evaluated as either inhibitors and/or substrates for the enzymes catechol O-methyltransferase, phenylethanolamine N-methyltransferase, histamine N-methyltransferase, and hydroxyindole O-methyltransferase. To evaluate these analogues as substrates for SAM-dependent methyltransferases, the corresponding methyl-14C compounds were prepared and tested for their abilities to donate their methyl group to the appropriate acceptor molecules. In addition, the unlabeled SAM analogues were tested for their inhibitory activities in these same transmethylation reactions. In general, it could be conlcuded from these studies that methyltransferases show very strict specificity for the structural features of SAM. This strict specificity holds for the enzymatic binding and methyl-donating abilities of this molecule. In fact, it could be concluded from the results of this study that methyltransferases show a higher specificity for the structural features of the substrate L-SAM than for the structural features of the product S-adenosyl-L-homocysteine (L-SAH).

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