Mechanism of Inhibition of Bovine Pineal Gland Hydroxyindole‐O‐Methyltransferase (EC 2.1.1.4) by Divalent Cations

Abstract

The effect of inhibitory cations on hydroxyindole-O-methyltransferase (HIOMT) was investigated in order to elucidate the mechanisms involved. Copper inhibition of HIOMT can be classified as partial, classic noncompetitive inhibition with respect to S-adenosyl methionine and as partial, mixed noncompetitive inhibition with respect to N-acetylserotonin. Binding of copper to the enzyme impaired but did not prevent catalysis, and the difference in inhibitory pattern observed with both substrates may be due to the obligatory order of substrate binding to HIOMT. Calcium and magnesium were uncompetitive inhibitors of HIOMT with respect to both substrates and binding of these cations to the enzyme prevents catalysis. The cations presumably bind to HIOMT in the vicinity of the catalytic site and in some way prevent or impair the catalytic process, possibly by interfering with substrate binding to the enzyme, interfering with methyl transfer or product release after catalysis, or a combination of these effects. The different inhibitory patterns observed may result from differences in molecular size or outer electron shell valence of the cations.

Probable mechanism of catalysis of pineal gland hydroxyindole-O-methyltransferase (HIOMT) from rainbow trout (Salmo gairdneri)

“…This catalytic mechanism is the same as that determined previously for bovine pineal HIOMT (Satake and Morton, 1979;Morton, 1986) and suggests that, although subtle differences may exist between species HIOMT's, there is species homogeneity in the catalytic mechanism.…”

“…As with the bovine enzyme (Morton, 1986), trout HIOMT was capable of methylating all the hydroxyindoles commonly found in the pineal gland (Table 1) which raises the possibility that methoxyindoles other than melatonin may be physiologically active particularly as these substances have been suggested to be bioactive (Mullen et al, 1979;Pevet, 1983;Reiter and Vaughan, 1977). Of particular interest was the observation that hydroxytryptophol had a similar affinity to the melatonin precursor, N-acetylserotonin, for trout HIOMT and it is possible, therefore, that methoxytryptophol may be physiologically important in the trout as a mediator of pineal activity.…”

“…Substrate specificities and catalytic mechanisms have been reported for mammalian HIOMT (Axelrod and Weissbach, 1961 ;Satake and Morton, 1979;Morton, 1986) and it was of interest to compare these results with those obtained from trout pineal HIOMT. A previous report has indicated that the assay systems routinely used fro mammalian HIOMT determination are adequate for estimation of teleost enzyme activity (Smith and Weber, 1973); these observations were further investigated in this study.…”

“…A previously described method (Morton, 1986) was used with modification. To each 25 gl aliquot of trout pineal homogenate was added 25 gl of S-adenosyl-L-l-methyI-~4C] methionine (specific activity 1.47 mCi/mmol; Amersham International, Bucks., U.K.),I25 gl of N-acetylserotonin (Sigma, St. Louis, MI) and 25 pl of 0.2 M phosphate buffer such that the desired concentration of substrates was obtained in a 0.05 M phosphate buffer pH 7,9 (unless otherwise specified).…”

Possible mechanisms of inhibition and activation of rat N-acetyltransferase (EC 2.3.1.5.) by cations

“…Although these reports all indicate that some type of relationship exists, no mechanisms have been proposed to explain the cation/pineal interactions. A recent reports showed that cations have differential effects on the activities of NAT and HIOMT in vitro (Morton, 1987a) and subsequently it was determined that copper was a partial, mixed noncompetitive inhibitor and calcium and magnesium were uncompetitive inhibitors of HIOMT (Morton. 1987b).…”