Inhibition of Glycine N-Methyltransferase by 5-Methyltetrahydrofolate Pentaglutamate

Abstract

Glycine N-methyltransferase (EC 2.1.1.20) catalyzes the methylation of glycine by S-adenosylmethionine to form sarcosine and S-adenosylhomocysteine. The enzyme was previously shown to be abundant in both the liver and pancreas of the rat, to consist of four identical monomers, and to contain tightly bound folate polyglutamates in vivo. We now report that the inhibition of glycine N-methyltransferase by (6S)-5-CH 3 -H 4 PteGlu 5 is noncompetitive with regard to both S-adenosylmethionine and glycine. The enzyme exhibits strong positive cooperativity with respect to S-adenosylmethionine. Cooperativity increases with increasing concentrations of 5-CH 3 -H 4 PteGlu 5 and is greater at physiological pH than at pH 9.0, the pH optimum. Under the same conditions, cooperativity is much greater for the pancreatic form of the enzyme. The V max for the liver form of the enzyme is approximately twice that of the pancreatic enzyme, while K m values for each substrate are similar in the liver and pancreatic enzymes. For the liver enzyme, at pH 7.0 half-maximal inhibition is seen at a concentration of about 0.2 M (6S)-5-CH 3 -H 4 PteGlu 5 , while at pH 9.0 this value is increased to about 1 M. For the liver form of the enzyme, 50% inhibition with respect to Sadenosylmethionine at pH 7.4 occurs at about 0.27 M. The dissociation constant, K s , obtained from binding data at pH 7.4 is 0.095. About 1 mol of (6S)-5-CH 3 -H 4 PteGlu 5 was bound per tetramer at pH 7.0, and 1.6 mol were bound at pH 9.0. The degree of binding and inhibition were closely parallel at each pH. At equal concentrations of (6R,6S)-and (6S)-5-CH 3 -H 4 PteGlu 5 , the natural (6S) form was about twice as inhibitory. These studies indicate that glycine N-methyltransferase is a highly allosteric enzyme, which is consistent with its role as a regulator of methyl group metabolism in both the liver and the pancreas.

Folate and homocysteine metabolisms and their roles in the biochemical basis of neuropsychiatry

“…Studies on cell cultures have shown that unbound or free folate concentrations are negligible (18). The major proteins that tightly bind intracellular folate in rat liver include mitochondrial sarcosine and dimethylglycine dehydrogenase, cytoplasmic glycine-N-methyltransferase (19), and 10-formyltetrahydrofolate dehydrogenase. These proteins are synthesized in large amounts in the liver, which stores almost half of the total body folate (20).…”

Acetylation of N-terminal valine of glycine N-methyltransferase affects enzyme inhibition by folate

“…Subtracting the free concentration of folate from the total concentration gave the concentration of bound folate (F bound ). When binding of folate pentaglutamate was studied the concentration of folate in the filtrate was measured by a fluorescence method with excitation at 297 nm and emission at 359 nm at pH 2.5 [14]. Fluorescence measurement was done on a Cary Eclipse Fluorescence Spectrophotometer (Varian, Inc.).…”

“…A number of previous studies have noted that GNMT from various sources display cooperativity with respect to AdoMet concentration in kinetic studies [12][13][14]. This cooperativity is increased in the presence of folate suggesting that some of the preparations that showed cooperativity may have had bound folate as a contaminant.…”

“…GNMT has been isolated and studied from a variety of sources [11][12][13]; inhibition of enzyme activity by folate was described in some detail [14] and several crystal structures were solved [15][16][17][18]. Recently the crystal structure of the GNMT-folate complex was reported [19].…”

The Many Flavors of Hyperhomocyst(e)inemia: Insights from Transgenic and Inhibitor-Based Mouse Models of Disrupted One-Carbon Metabolism

“…In a wild-type mouse after overnight fasting, the [AdoMet]:[AdoHcy] ratio was 0.9 in liver and 47 in heart (13). Glycine N-methyltransferase activity is inhibited by methyltetrahydrofolate and activated by AdoMet (71). Thus, in an Mthfr-deficient mouse, with extremely low levels of methyltetrahydrofolate, glycine Nmethyltransferase should be maximally active in the liver, while in a methionine synthase reductase-deficient mouse or a methionine synthase-deficient mouse, glycine N-methyltransferase would be expected to be substantially inhibited in liver, resulting in a sparing of the [AdoMet]:[AdoHcy] ratio, and perhaps, in the case of methyl trapping, even leading to an increase in the [AdoMet]:[AdoHcy] ratio.…”