VARIATIONS IN AROMATIC AMINO ACID DECARBOXYLASE ACTIVITY TOWARDS DOPA AND 5‐HYDROXYTRYPTOPHAN CAUSED BY pH CHANGES AND DENATURATION
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Abstract —DOPA and 5‐hydroxytryptophan (5‐HTP) are generally supposed to be decarboxylated in mammalian tissues by a single enzyme, the two activities being present in constant ratio through a variety of purification procedures. It has now been shown that the ratio of activity of the liver enzyme towards the two substrates can be altered by mild treatments, such as might be used in solubilization of brain preparations. DOPA decarboxylase activity was preferentially inactivated by sodium dodecyl sulphate treatment, and 5‐HTP decarboxylation by urea. Previous reports that the two substrates show different pH optima but are mutually competitive, have been confirmed. The K m of the enzyme towards 5‐HTP was lowest at pH 7.8 (the optimum pH for decarboxylation of this amino acid), but the variation with pH of the K m towards DOPA was unrelated to the pH optimum for decarboxylation. There appeared to be no relation between the probable ionization state of the substrates and the pH dependence of the enzyme. Studies on the binding characteristics of the enzyme for the two products, dopamine and serotonin, did not show any specific saturable binding. It is proposed that the enzyme has a complex active site, with separate affinity sites for the two substrates, adjacent to a single catalytic site.Keywords:
Decarboxylation
5-Hydroxytryptophan
Abstract 5‐Hydroxytryptophan has been found to be beneficial when administered alone or in combination with extracerebral aromatic amino acid decarboxylase inhibitors for therapeutic purposes in various disorders in which myoclonus is prominent. In five subjects the effect of decarboxylase inhibitors on the accumulation and elimination of 5‐hydroxytryptophan in plasma was studied. The plasma concentrations of 5‐hydroxytryptophan were increased about ten fold by pretreatment with the decarboxylase inhibitors, carbidopa and benserazide. Half‐lives of 2.2‐3.0 hrs. were obtained following oral administration of a single dose of 5‐hydroxytryptophan with or without pretreatment. In one subject the half‐life of 5 hydroxytryptophan in plasma increased to 5.5 hrs. during long‐term treatment with carbidopa.
Benserazide
Carbidopa
5-Hydroxytryptophan
Decarboxylase inhibitor
Plasma levels
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Decarboxylation
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Abstract —DOPA and 5‐hydroxytryptophan (5‐HTP) are generally supposed to be decarboxylated in mammalian tissues by a single enzyme, the two activities being present in constant ratio through a variety of purification procedures. It has now been shown that the ratio of activity of the liver enzyme towards the two substrates can be altered by mild treatments, such as might be used in solubilization of brain preparations. DOPA decarboxylase activity was preferentially inactivated by sodium dodecyl sulphate treatment, and 5‐HTP decarboxylation by urea. Previous reports that the two substrates show different pH optima but are mutually competitive, have been confirmed. The K m of the enzyme towards 5‐HTP was lowest at pH 7.8 (the optimum pH for decarboxylation of this amino acid), but the variation with pH of the K m towards DOPA was unrelated to the pH optimum for decarboxylation. There appeared to be no relation between the probable ionization state of the substrates and the pH dependence of the enzyme. Studies on the binding characteristics of the enzyme for the two products, dopamine and serotonin, did not show any specific saturable binding. It is proposed that the enzyme has a complex active site, with separate affinity sites for the two substrates, adjacent to a single catalytic site.
Decarboxylation
5-Hydroxytryptophan
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Decarboxylation
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Amino acid decarboxylation activity in dispersed rat pancreas acinar cells and fractions derived by differential centrifugation of homogenate of these cells was studied. The rate of decarboxylation was measured by determining the rate of production of the [3H]-amine from [3H]-amino acid or the rate of production of 14C(>2 from the [14C]-carboxy-labelled amino acid. Only the hydroxylated amino acids L-dopa and 5-hydroxytryptophan are decarboxylated by intact dispersed pancreas acinar cells or cell homogenates at all pH values and amino acid concentrations tested. The decarboxylase activity is located exclusively in the cell cytosol. Each substrate competitively inhibits the decarboxylation of the other and the decarboxylation of each is inhibited by NSD-1055. The estimated Km and Vmax are, for L-dopa, 4.8 × 10–5M and 2.5 nmol/mg protein/min and for 5-hydroxytryptophan, 2.9 × 10–5M and 0.3 nmol/mg protein/min. The pH optimum for 5-hydroxytryptophan decarboxylation is from 7.0–8.5 while that for L-dopa is 7.0. It is concluded that pancreas acinar cells possess a single aromatic amino acid decarboxylase specific for the hydroxylated amino acids L-dopa and 5-hydroxytryptophan, and which is similar in all properties studied to the aromatic amino acid decarboxylase found in several other mammalian tissues.
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5-Hydroxytryptophan
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We measured decarboxylation of oral L-dopa in patients chronically treated with L-dopa, and in untreated controls. Chronic L-dopa and carbidopa administration did not affect the extent of whole-body decarboxylation, and it is therefore unlikely that on-off fluctuations are related to chronic changes in the activity of L-aromatic amino acid decarboxylase. The observed duration of action and dose-response properties of carbidopa suggested that current empirically based dose schedules are optimal and supported the concept that decarboxylase inhibitors enhance the clinical effect of L-dopa largely by reducing the extent of first-pass metabolism rather than through an action on the decarboxylase enzyme in cerebral capillaries.
Decarboxylation
Carbidopa
Decarboxylase inhibitor
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Tryptophan hydroxylase
5-Hydroxytryptophan
Limiting
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Decarboxylation
Dihydroxyphenylalanine
Pyridoxal phosphate
Pyridoxal 5-Phosphate
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The effects of L-5-hydroxytryptophan (L-5HTP) and L-5HTP inosinate injection on brain 5-hydroxyindoles in rats were compared. L-5HTP and L-5HTP inosinate caused indistinguishable dose-dependent increases in 5HTP and 5HIAA (5-hydroxyindoleacetic acid) but not serotonin concentrations in whole brain at 1 hr in rats. Our results do not substantiate a previous claim that L-5HTP inosinate is superior to L-5HTP itself in increasing brain serotonin formation.
5-Hydroxytryptophan
5-Hydroxyindoleacetic acid
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Abstract DL‐ x ‐Difluoromethyl DOPA (DFMD, RMI 71801), an enzyme‐activated irreversible inhibitor of aromatic L‐amino acid decarboxylase in vitro , produces a rapid, long‐lasting and dose‐dependent inhibition of aromatic L‐amino acid decarboxylase in peripheral tissues of mice when administered i.p. or orally. Doses of 500 mg/kg i.p. produce only very slight inhibition of the enzyme activity in mouse brain whilst inhibiting the enzyme activity of peripheral tissues by more than 90%. With L‐[ 3 H]‐DOPA co‐administration brain concentrations of L‐[ 3 H]DOPA and 3 H‐catecholamines are increased 3‐ to 8‐fold concomitant with a decrease in the peripheral decarboxylation of L‐[ 3 H]DOPA. Under these conditions it is clear that the slight inhibition of enzyme activity in the brain is totally inadequate to inhibit the decarboxylation of L‐DOPA in this organ. Similarly, the decarboxylation of exogenously supplied 5‐hydroxytryptophan is inhibited peripherally with a consequent increase in brain serotonin concentrations. DFMD is another example of an enzyme‐activated irreversible inhibitor which due to its novel and specific mechanism of action, may offer advantages over existing decarboxylase inhibitors.
Decarboxylation
Decarboxylase inhibitor
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