Tyrosine hydroxylase is an iron-containing monooxygenase that uses a tetrahydropterin to catalyze the hydroxylation of tyrosine to dihydroxyphenylalanine in catecholamine biosynthesis. The role of the iron in this enzyme is not understood. Purification of recombinant rat tyrosine hydroxylase containing 0.5-0.7 iron atoms/ subunit and lacking bound catecholamine has permitted studies of the redox states of the resting enzyme and the enzyme during catalysis. As isolated, the iron is in the ferric form. Dithionite or 6-methyltetrahydropterin can reduce the iron to the ferrous form. Reduction by 6-methyltetrahydropterin consumes 0.5 nmol/nmol of enzyme-bound iron, producing quinonoid 6-methyldihydropterin as the only detectable product. In the presence of oxygen, reoxidation to ferric iron occurs. During turnover the enzyme is in the ferrous form. However, a fraction is oxidized during turnover; this can be trapped by added catechol or by the dihydroxyphenylalanine formed during turnover.Tyrosine hydroxylase catalyzes the hydroxylation of tyrosine to form dihydroxyphenylalanine (DOPA), 1 the rate-limiting step in the biosynthesis of the catecholamine neurotransmitters (1). This is one of a small family of tetrahydropterinutilizing monooxygenases found in the central nervous system; the others are phenylalanine hydroxylase and tryptophan hydroxylase. The mechanisms of these enzymes are very poorly understood, and there are as yet no structures available. Tyrosine hydroxylase has been known for some time to contain 1 iron atom/subunit (2). Studies of the metal dependence of the catalytic activity have shown that ferrous iron is required; no other metal has been found to be catalytically active (3, 4). The iron atom is bound to amino acid side chains rather than a porphyrin ring; recently two of the metal ligands in rat tyrosine hydroxylase have been identified as histidines 331 and 336 (5). As yet, the role of the iron in tyrosine hydroxylase remains unclear. However, the lack of activity of iron-depleted enzyme (3, 4), or of enzyme in which a metal ligand has been modified by site-directed mutagenesis (5), is consistent with an essential role in catalysis. In addition, NMR measurements have shown that the amino acid substrate binds close to the iron, placing the metal in the active site (6).Until relatively recently, studies of the metal site in tyrosine hydroxylase were hindered by the difficulties of obtaining sufficient amounts of purified enzyme for study. Within the last decade preparations from bovine adrenal medulla (7) and rat pheochromocytoma (8) have permitted physical studies. The enzyme from both sources contained 0.6 -0.7 iron atoms/subunit. EPR spectroscopy showed that the iron was in the iron-(III) state when isolated. Furthermore, the enzyme had a bluegreen color due to the presence of tightly bound catecholamines interacting with the metal (8, 9). More recently, several laboratories have successfully expressed human or rat tyrosine hydroxylase in bacteria, providing access to significantly more mater...

The effects of phosphorylation at Ser40 of rat tyrosine hydroxylase on the affinities of catechols have been determined with both the ferric and ferrous forms of the enzyme. Phosphorylation had no effect on the Ki value for the inhibition of the ferrous enzyme by either dopamine or DOPA when the initial rate of turnover was measured in assays. However, phosphorylation of the ferric enzyme resulted in a 17-fold decrease in affinity for DOPA and a 300-fold decrease in the affinity for dopamine, while the affinity for dihydroxynaphthalene was unchanged. The changes in binding affinity for the two catecholamines were almost exclusively due to large increases in the dissociation rate constants upon phosphorylation. These results support a novel mechanism for regulation in which phosphorylation affects binding of catecholamines to the catalytically inactive ferric form of the tyrosine hydroxylase.

Chemical reactions between the isothiazolone biocides, N-methylisothiazol-3-one (MIT), benzisothiazol-3-one (BIT) and 5-chloro-N-methylisothiazol-3-one (CMIT) with cysteine have been investigated by u.v. and NMR spectroscopy. At physiological pH all three agents interacted oxidatively with thiols to form disulphides. Further interaction with thiols caused the release of cystine and formation of a reduced, ring-opened form of the biocide (mercaptoacrylamide). In an analogous fashion to the initial reaction the mercaptoacrylamide reacted with another molecule of biocide to give biocide dimers. NMR spectral studies indicated that for CMIT the mercaptoacrylamide form is capable of tautomerization to a highly reactive thio-acyl chloride. Formation of mercaptoacrylamide was in all cases highly pH-dependent. Alcohol dehydrogenase was insensitive to all three agents but was highly sensitive to CMIT when co-administered with dithiothreitol. Capacity to form a thioacyl chloride from the mercaptoacrylamide is suggested to account for much of this enhanced activity. Stopped-flow spectroscopic studies showed rates of reaction with glutathione (GSH) to directly parallel antimicrobial activity. Additionally, CMIT was able to react directly with both ionization states of GSH (pH 7-10) whilst BIT and MIT appeared only to interact when the glutamyl-nitrogen of GSH was charged (pH 8.5).

The protein serine/threonine phosphatase designated PP5 has little basal activity, and physiological activators of the enzyme have never been identified. Purified PP5 can, however, be activated by partial proteolysis or by the binding of supraphysiological concentrations of polyunsaturated long-chain fatty acids to its tetratricopeptide repeat (TPR) domain. To test whether activation of PP5 by polyunsaturated but not saturated fatty acids was an artifact of the lower solubility of saturated fatty acids, the effects of fatty acyl-CoA esters were examined. Saturated and unsaturated long-chain fatty acids are both freely water-soluble when esterified to CoA. Long-chain fatty acyl-CoA esters activated PP5 at physiological concentrations, with the saturated compounds being more effective. We investigated the effects of chain length and of the CoA moiety on PP5 activation. Chains of 16 carbons or more were required for optimal activation, with no activation observed below 10 carbons. On the basis of competition studies using acetyl-CoA, the function of the CoA moiety appeared to be to increase solubility of the fatty acyl moiety rather than to interact with a specific binding site. These data suggested that long-chain fatty acid-CoA esters might be physiological activators of PP5 and point to a potential link between fatty acid metabolism and signal transduction via this enzyme. Because heat shock protein 90 is also known to bind to the TPR domain of PP5 via its C-terminal domain (C90), we investigated its effect on PP5 activity. C90 activated the enzyme approximately 10-fold. Thus, we have identified two potential physiological activators of PP5.

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