Synthetic decarboxylated S-adenosyl-L-methionine (dcAdoMet), a mixture of the absolute configuration of S and R at the sulfonium center, was evaluated as a substrate for the measurement of spermidine synthase activity. The diastereomers were separated by HPLC with an isocratic elution, and the constant for racemization at the sulfur was determined to be 2.4x10(-6) s(-1) at 37 degrees C and pH 1.5 for the first-eluted biologically active isomer (S-dcAdoMet) and 2.0x10(-6) s(-1) for the second-eluted biologically inactive isomer (R-dcAdoMet). The peak area ratio of S-dcAdoMet to R-dcAdoMet of 48 to 52 in HPLC supported the different racemization constants. Similar substrate activity of dcAdoMet to that of S-dcAdoMet was demonstrated by enzymatic spermidine synthesis. It was shown from the result that the racemized [methyl-(14)C]dcAdoMet prepared in this report was useful for measuring spermidine synthase activity.
Spermidine synthase (spd syn, putrescine aminopropyltransferase) has been shown to catalyze the transfer reaction of the aminopropyl moiety of decarboxylated S-adenosyl-Lmethionine (dcAdoMet) into putrescine in the formation of spermidine, which is widely distributed among living organisms with increasing levels related to cell growth. [1][2][3] In addition to the reported amino acid sequence of spd syn from various sources, 4,5) the crystal structures of some of these bacterial enzymes have been recently reported 6) (PDB accession codes 1INL, 1JQ3, 1MJF, 1IY9, and 1UIR). One of the significant differences between the amino acid sequence of mammalian and bacterial enzymes is the content of cysteine residue, i.e. ten residues for mammalian enzymes, three for Thermotoga maritima, and none for Bacillus subtilis and Pyrococcus furiosus. Accordingly, the determination of each cysteine residue as either sulfhydryl or disulfide is essential for the accurate homology modeling of mammalian spd syn. The primary sequence of rat spd syn is shown in Fig. 1. 7)The use of specific inhibitors is another approach in the study of the substrate-binding site. Using a number of monoamine and diamine compounds, we have proposed a model of the putrescine-binding site of pig spd syn, 8) which features a relatively large hydrophobic cavity adjacent to a negatively charged site. Presumably, one of the amino group of putrescine is protonated and binds to this charged site, and the other amino group is not protonated and binds to the hydrophobic cavity, to be aminopropylated by dcAdoMet. The substrate-binding site of our model shows good agreement with that of the crystal structure of the T. maritima enzyme.6)The present study was undertaken to identify the ten cysteine residues of rat spd syn exist as sulfhydryl or disulfide, and to investigate the putrescine-binding site using several compounds that are based on two known potent inhibitors for mammalian spd syn, trans-4-methylcyclohexylamine (4MCHA) and n-butylamine (BA). Based on the results, a three-dimensional model for mammalian spd syn was proposed using the homology modeling approach based on the crystal structure of the T. maritima enzyme. MATERIALS AND METHODSChemicals Dithiothreitol (DTT), guanidine hydrochloride, monoiodoacetic acid (MIA), trans-4-hydroxycyclohexyl amine (4HCHA), 5-[N-(iodoacetamidoethyl)amino]naphthalene-1-sulfonic acid (IAEDANS), 2-nitro-5-thiocyanobenzoic acid (NTCB), and tris(2-carboxyethyl)phosphine hydrochloride (TCEP) were purchased from Sigma. Decarboxylated S-adenosylmethionine (dcAdoMet) was prepared in our laboratories. 9) N-Hydroxysuccinimidyl bromoacetate (HSBA) was also prepared in our laboratories using bromoacetic acid, N-hydroxysuccinimide, and dicyclohexylcarbodiimide. n-Propylamine, n-butylamine (BA), n-pentyl amine (PA), n-hexylamine (HA), and cyclohexylamine (CHA) were purchased from Tokyo Kasei Kogyo Co. Ltd., and were recrystallized as their hydrochlorides. 4-Methylcyclohexylamine (as a mixture of cis-and trans-isomers) was purchased fro...
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