S-Adenosylmethionine:protein-arginine methyltransferase. Purification and mechanism of the enzyme

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Abstract
Protein methylase I (S-adenosylmethionine: protein-arginine methyltransferase, EC 2.1.1.23) was purified from calf brain approximately 120-fold with a 14% yield. The final preparation is completely free of any other protein-specific methyltransferases and endogenous substrate protein. The enzyme has an optimum pH of 7.2 and pI [isoelectric point] value of 5.1. The Km values for S-adenosyl-L-methionine, histone H4 and an ancephalitogenic basic protein are 7.6 .times. 10-6, 2.5 .times. 10-5 and 7.1 .times. 10-5 M, respectively, and the Ki value for S-adenosyl-L-homocysteine is 2.62 .times. 10-6 M. The enzyme is highly specific for the arginine residues of protein, and the end products after hydrolysis of the methylated protein are NG,NG-di(asymmetric), NG,N''G-di(symmetric), and NG-monomethylarginine. The ratio of [14C]methyl incorporation into these derivatives by enzyme preparation at varying stages of purification remains unchanged at 40:5:55, strongly indicating that a single enzyme is involved in the synthesis of the 3 arginine derivatives. The kinetic mechanism of the protein methylase I reaction was studied with the purified enzyme. Initial velocity patterns converging at a point on the extended axis of abscissas were obtained with either histone H4 or S-adenosyl-L-methionine as the varied substrate. Product inhibition by S-adenosyl-L-homocysteine with S-adenosyl-L-methionine as the varied substrate was competitive regardless of whether or not the enzyme was saturated with histone H4. When histone H4 is the variable substrate, noncompetitive inhibition was obtained with S-adenosyl-L-homocysteine under conditions where the enzyme is not saturated with the other substrate, S-adenosyl-Lmethionine. The mechanism of the protein methylase I reaction is probably a Sequential Ordered Bi Bi mechanism with S-adenosyl-L-methionine as the 1st substrate, histone H4 as the 2nd substrate, methylated histone H4 as the 1st product and S-adenosyl-L-homocysteine as the 2nd product released.
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