Abstract
Methionine adenosyltransferase (MAT) catalyzes the adenosine 5′-triphosphate (ATP) and l-methionine (l-Met) dependent formation of S-adenosyl-l-methionine (SAM), the principal methyl donor of most biological transmethylation reactions. We carried out in-depth kinetic studies to further understand its mechanism and interaction with a potential regulator, Mat2B. The initial velocity pattern and results of product inhibition by SAM, phosphate, and pyrophosphate, and dead-end inhibition by the l-Met analog cycloleucine (l-cLeu) suggest that Mat2A follows a strictly ordered kinetic mechanism where ATP binds before l-Met and with SAM released prior to random release of phosphate and pyrophosphate. Isothermal titration calorimetry (ITC) showed binding of ATP to Mat2A with a Kd of 80 ± 30 μM, which is close to the Km(ATP) of 50 ± 10 μM. In contrast, l-Met or l-cLeu showed no binding to Mat2A in the absence of ATP; however, binding to l-cLeu was observed in the presence of ATP. The ITC results are fully consistent with the product and dead-inhibition results obtained. We also carried out kinetic studies in the presence of the physiological regulator Mat2B. Under conditions where all Mat2A is found in complex with Mat2B, no significant change in the kinetic parameters was observed despite confirmation of a very high binding affinity of Mat2A to Mat2B (Kd of 6 ± 1 nM). Finally, we found that while Mat2A is unstable at low concentrations (<100 nM), rapidly losing activity at 37 °C, it retained full activity for at least 2 h when Mat2B was present at the known 2:1 Mat2A/Mat2B stoichiometry.
Highlights
S-Adenosyl-L-methionine (SAM) is the principal methyl donor in biology and as such is an essential metabolite required for all forms of life
Mat2A appears relatively slow to maintain the metabolic supply of SAM; the kcat agrees well with values reported by other groups using different assay methods (0.27 and 0.35 s−1)[10,21] and is remarkably within 2-fold[1] to that reported using native Mat2A/Mat2B purified from human lymphocytes.[19,20]
It would be important to measure the absolute concentration of Mat2A in different cell types under different physiological conditions to determine if the in vitro rate of Mat2A is fast enough to maintain the metabolic supply of SAM
Summary
S-Adenosyl-L-methionine (SAM) is the principal methyl donor in biology and as such is an essential metabolite required for all forms of life. SAM is synthesized by methionine adenosyltransferase (Mat; EC 2.1.5.6) using adenosine 5′-triphosphate (ATP) and L-methionine (L-Met) as substrates generating SAM, inorganic phosphate (Pi), and pyrophosphate (PPi) as products.[8,9] Humans possess two Mat isozymes, Mat1A and Mat2A, which are encoded by the mat1a and mat2a genes, respectively. These enzymes are highly similar, sharing 84% primary sequence identity and 93% sequence similarity, and have nearly identical structures (RMSD
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