Chromatographic analysis of the chiral and covalent instability of S-adenosyl-L-methionine

Abstract

The chirality of biologically active S-adenosyl-L-methionine (AdoMet) is S,S, where the designations refer to the sulfur and the alpha-carbon, respectively. This paper describes a cation-exchange high-performance liquid chromatographic (HPLC) method for separating (S,S)-AdoMet from the biologically inactive (R,S)-AdoMet that results from racemization at the sulfur. This method was used to measure the rates of the degradation reactions of (S,S)-AdoMet as a function of pH. These reactions and the first-order rate constants, which were found at 37 degrees C and pH 7.5, are racemization, 1.8 X 10(-6) s-1; cleavage to homoserine lactone and 5'-(methylthio)adenosine, 4.6 X 10(-6) s-1; and hydrolysis to adenine and S-pentosylmethionine, 3 X 10(-6) s-1. Racemization showed no change in rate over the pH range from 7.5 to 1.5. The cleavage reaction persisted until the pH was lowered to 1.5, but hydrolysis ceased at pH 6. Commercial samples of nonradioactive AdoMet contained 20-30% (R,S)-AdoMet, while a sample of [methyl-3H]AdoMet had less than 1% (R,S)-AdoMet. Preparing enzyme substrates by mixing such samples will cause an underestimate of specific activity and an overestimate of the amount of product. The (R,S)-AdoMet/(S,S)-AdoMet ratio in mouse liver was 0.03, much less than the value of 0.19 calculated from the above rate constants. An enzyme extract from mouse liver did not degrade (R,S)-AdoMet, but a more thorough search may find such an activity. In any event, the cleavage and hydrolysis reactions partially balance the racemization of (S,S)-AdoMet in vivo and prevent excessive accumulation of (R,S)-AdoMet.

Cell division, one-carbon metabolism and methionine synthesis in a metK-deficient Escherichia coli mutant, and a role for MmuM

“…Hoffman presented a surprising variety of such values in different preparations, from Sigma 20.7 % to Boehringer Mannheim 31.4 % (Hoffman, 1986). A high value of 47-50 % (R)-SAM (approximately the equilibrium value) was reported for SAM-e purified as in this work (Cannon et al, 2002).…”

Expression of Secreted His-Tagged S-adenosylmethionine Synthetase in the Methylotrophic Yeast Pichia pastoris and Its Characterization, One-Step Purification, and Immobilization

“…The optimal pH value of the endogenous SAM synthetase is near pH 7.6 (29), while the value of the recombinant synthetase is pH 8.5. However, the product SAM is slowly decomposed in neutral or alkaline solution at room temperature (30). It is therefore necessary to acidify the reaction mixture with HClO 4 in order to stop the reaction and stabilize the product.…”

Enzymatic Properties of S-Adenosylmethionine Synthetase from the Archaeon Methanococcus jannaschii

“…The thermal lability of AdoMet, both in chirality at the sulfonium center and in covalent bonding (34), requires a substantial metabolic flux through the synthetic reaction to maintain a pool of substrate for further metabolic requirements. The larger apparent activation energy for the MjMAT in the 0 -37°C range reflects an increase in the apparent enthalpy of activation, whereas there is a more favorable entropy of activation.…”