Tetrahydropteridine cofactor

The rate-limiting step in the synthesis of the catecholamines from tyrosine is tyrosine hydroxylase, so that any drug or substance which can reduce the activity of this enzyme, for example by reducing the concentration of the tetrahydropteridine cofactor, will reduce the rate of synthesis of the catecholamines. Under normal conditions tyrosine hydroxylase is maximally active, which implies that the rate of synthesis of the catecholamines is not in any way dependent on the dietary precursor tyrosine. Catecholamine synthesis may be reduced by end product inhibition. This is a process whereby catecholamine present in the synaptic cleft, for example as a result of excessive nerve stimulation, will reduce the affinity of the pteridine cofactor for tyrosine hydroxylase and thereby reduce synthesis of the transmitter. The experimental drug alpha-methyl-para-tyrosine inhibits the rate-limiting step by acting as a false substrate for the enzyme, the net result being a reduction in the catecholamine concentrations in both the central and peripheral nervous systems. 

Tyrosine is the immediate precursor of catecholamines, and tyrosine hydroxylase is the rate-limiting enzyme in catecholamine biosynthesis. Tyrosine hydroxylase is found in both soluble and particle-bound forms only in tissues that synthesize catecholamines it functions as an oxidoreductase, with tetrahydropteridine as a cofactor, to convert L-tyrosine to L-dihydroxyphenylalanine (L-dopa). 

Figure 2.16. Pathways for the synthesis and metabolism of the catecholamines. A=phenylalanine hydroxylase+pteridine cofactor+02 B=tyrosine hydroxylase+ tetrahydropteridine+Fe+++02 C=dopa decarboxylase+pyridoxal phosphate D= dopamine beta-oxidase+ascorbate phosphate+Cu+++02 E=phenylethanolamine N-methyltransferase+S-adenosylmethionine l=monoamine oxidase and aldehyde dehydrogenase 2=catechol-0-methyltransferase+S-adenosylmethionine.

Oxidative cleavage of the O-alkyl linkage in glycerolipids is catalyzed by a microsomal tetrahydropteridine (Pte-H4)-dependent alkyl monooxygenase (Fig. 12) (T.-C. Lee, 1981). The required cofactor, Pte H4, is regenerated from Pte-Hj by an NADPH-linked pteridine reductase, a cytosolic enzyme. Oxidative attack on the ether bond in lipids is similar to the enzymatic mechanism described for the hydroxylation of phenylalanine. Fatty aldehydes produced via the cleavage reaction can be either oxidized to the corresponding acid or reduced to the alcohol by appropriate enzymes. 

Reactions 2, 3, and 4 tell us little about how oxygen is activated during the hydroxylation reaction, and at the moment one can only speculate about the details. The scheme and the results on which it is based do, however, rule out several general types of hydroxylation mechanism. The fact that the enzyme can be reduced anaerobically by ascorbate to a form which actively supports substrate hydroxylation in the absence of ascorbate rules out any mechanism for this enzyme-catalyzed reaction in which the ascorbate functions as an oxygen carrier. Such a role has been postulated for tetrahydropteridines (id), which can serve as specific electron-donating cofactors (just as ascorbate does with dopamine )S-hydroxylase) in certain aromatic hydroxylation reactions (iO, 12). 

The unknown cofactor could be replaced by tetrahydrofolic acid and by other tetrahydropteridines, e.g., 2-amino-4-hydroxy-6,7-dimethyl-tetrahydropteridine. The latter is convenient to use because it is more stable than tetrahydrofolic acid. Interchanging the 2-amino-4-hydroxy groups of the natural pteridines caused loss of coenzyme activity... 

In contrast to the activity of the synthetic tetrahydropteridines, the cofactor purified from rat liver is not active in the defluorination reaction. It is not known whether this is a reflection of a fundamental difference in the mode of action of the cofactor as compared with the synthetic pteridines or whether it is related to the much smaller amounts of cofactor which can be used in this type of experiment. 

This kind of mechanism also has the advantage of providing an explanation for the inactivity of the natural cofactor in the defluorination reaction without discarding the idea that its mode of action is essentially the same as that of the synthetic tetrahydropteridines. According... 

The general characteristics of the hydroxylation reaction appear to be the same in the presence of synthetic tetrahydropteridines and the natural cofactor. There are, however, differences in the behavior of the two types of compound. These have already been mentioned but it might be useful to summarize them here. 

The time course of tyrosine formation is different, the reaction with the cofactor being characterized by a pronounced lag period which is not ordinarily seen with the tetrahydropteridines. In all probability, this difference is a reflection of the fact that the cofactor as isolated is not in the reduced, active form, although it may be partially reduced. 

The factor in glucose dehydrogenase preparations which can stimulate tyrosine formation can be replaced by crystalline catalase when tetrahydropteridines are used while catalase cannot replace glucose dehydrogenase with the cofactor. It is possible that the glucose dehydrogenase fractions contain another factor, besides catalase, which is required for the activity of the cofactor. This additional factor may also be involved in the conversion of the cofactor to an active form. 

Reducing agents such as 2-mercaptoethanol can partially replace TPNH when tetrahydropteridines are used but this is not true for the cofactor. 

The reductive reaction involving TPNH and the primary oxidation product of the tetrahydropteridine can proceed in the absence of sheep liver enzyme. The TPNH-cofactor reaction, on the other hand, shows an absolute requirement for the sheep liver enzyme. 

The cofactor, in contrast to tetrahydropteridines, is inactive in catalyzing the conversion of 4-fluorophenylalanine to tyrosine. 

These differences when viewed isolated from other facts seem to suggest that the natural cofactor is not a pteridine. An alternate view is that they (especially items 3, 4, and 5) point to a lowered reactivity of the cofactor as compared to the synthetic tetrahydropteridines which have been used. Such a lowered reactivity could be the result of a substitution on the pteridine ring. Until more is known about the structure of the cofactor, the significance of these differences cannot be fully evaluated. They do not, however, necessarily constitute evidence against a pteridine structure for the cofactor. 

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