Formylase

Equation 1 is catalyzed by glycinamide ribotide (GAR) trans-formylase and Equation 2 is catalyzed by aminoimidazole-carboxamide ribotide (AICAR) transformylase. 

This enzyme [EC 2.1.2.3], also known as AfCAR trans-formylase, catalyzes the reaction of 10-formyltetrahy-drofolate with 5 -phosphoribosyl-5-amino-4-imidazole-... 

A class of enzymes that was made accessible only recently is the peptide de-formylases. It was demonstrated that peptide deformylase can be used for kinetic resolutions, but they can also be employed to cleave off formyl protection groups [99]. Due to the stereoselectivity of the enzyme the enantiopurity of the product is also improved during the deprotection (Scheme 6.31). 

Changes in glucose-6-phosphatase, forminoglutamic acid transferase, urocanase, formylase, methyl-H4-folate dehydrogenase, histidiase, glutamic oxalacetic transaminase and carbonyl transferase and ornithine... 

The enzyme called formylase by Knox and Mehler (490, 591) and ky-nurenine formamidase by Jakoby (437) is present in liver in a considerable excess relative to tryptophan peroxidase-oxidase (e.g., 491), and formylky-nurenine is therefore not normally found in tissues or excreted in urine (e.g., 171). Partially purified tryptophan peroxidase-oxidase, from which formylase activity has been removed, accumulates formylkynurenine, shown (591) to be identical with synthetic (947 or better, 172) material. Formylase occurs widely in bacteria, and has been partially purified from Neurospora (437). In both higher and lower organisms the enzyme shows considerable specificity. 

Methionine is linked to these two kinds of tRNAs by the same aminoacyl-iRNA synthetase. A specific enzyme then formylates the amino group of methionine attached to tRNAf (Figure 30.17), The activated formyl donor in this reaction is N hformyltetrahydrofolate (p. 689). It is significant that free methionine and methionyl-tRNA., are not substrates for this trans-formylase. 

Homocysteine methyl transferase and glycinamide ribonucleotide (GAR) trans-formylase are examples of enzymes that require THF-coenzymes. 

It is not known exactly how methotrexate produces its effects in rheumatoid arthritis, but one theory is that it possibly increases levels of adenosine by blocking a step in purine biosynthesis, leading to accumulation of adenosine, which results in anti-inflammatory effects. It also inhibits the enzyme 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) trans-formylase, raising levels of AICAR, which in turn increases adenosine levels. It may also contribute to the phosphorylation of adenosine nucleotides creating an accumulation of adenosine in tissues. Caffeine is an adenosine receptor antagonist and therefore could reverse the effects of methotrexate. 

Kynurenine Formylase. Hydrolysis of formylkynurenine to kynurenine is catalyzed in liver preparations by an enzyme (formylase or kynurenine formamidase) with rather low specificity for aromatic formamides. The reaction with formylkynurenine, however, is faster than that with any analogs. A very similar enzyme has been obtained from Neurospora. In contrast, an enzyme from insects does not hydrolyze formylanthranilic acid, a model substrate for formylase from other sources, or other analo-... 

Formylase can be readily separated from the rest of the enzyme system, and when this is done formylkynurenine accumulates. This enzyme specifically hydrolyzes aromatic formamides. The highest activ-... 

The enzymes catalyzing each reaction were purified FH -formylase was crystallized (Rabinowitz and Pricer, 1958) and formimino-FH4 isolated and crystallized. Reaction (VI, e), as noted, is a new ATP-generating reaction. 

Protein synthesis begins with the N-terminal amino acid and proceeds from this point. In some bacteria, yeast, and higher organisms, this first aminoacyl-tRNA is known to be iV-formylmethionyl-tRNA Formylation of the amino function can be considered as a protecting group to prevent participation of the amino function is peptide bond formation. The fMet-tRNA is then the first aminoacyl-tRNA to bind to the ribosome and mRNA. After the protein is synthesized, the formyl group is removed by enzymatic cleavage (formylase). 

When formiminoglycine (FIG) was broken down to glycine, derivatives of folic acid were involved and ATP formation occurred (H, 297). These reactions are shown in Fig. 17. The first of these reactions was catalyzed by FIG formimino transferase FIG reacted with FH4, free glycine was formed, and iV -formimino-FH now carried the formimino grouping. In the second step, NH3 was released from iNT -formimino-FEU and the cyclic iVs. Vio-imidazolinium derivative of iV -formyl-FH4 resulted. The latter compound was also called lV ,Ari >-methenyl-FH4, anhydroleucovorin, or anhydrocitrovorum factor the enzyme responsible for the formation of the imidazolinium derivative from A -formimino-FH4 was named formimino-FH4 cyclodeaminase. In the third reaction, methenyl-FH4 cyclohydrolase catalyzed the conversion of lV ,iW -methenyl-FH4 to JV -formyl-FH4. The fourth step, which resulted in ATP formation, was catalyzed by FH4-formylase. ADP and P reacted with JW -formyl-FH4 and produced FH4, free formic acid, and ATP. The reverse of this reaction resulted in the activation of free formic acid [Eq. (5)]. 

In a similar way the purine antimetabolite 6-mercapto-purine (6-MP)/ after conversion to the monophosphoribo-tide, acts as an inhibitor of this enzyme (9.10). There have been described other sites of action of 6-MP (4-7) which are marked by arrows in figure 1. We have previously demonstrated the inhibition of the enzymic formate activation (tetrahydrofolate formylase) in leukemic cells by 6-MP (12). inspite of a rather high inhibitory concentration of 6-MP between 10 and 10 M, this inhibition has some practical clinical implications for the treatment of acute leukemia/ as it is detected in sensitive leukemic cells only. In accordance with reports of several authors (8,9/10) we have postulated/ that 6-MP has to be converted into 6-thioinosinic acid for exerting its inhibitory effect on the de novo synthesis of purine-nucleotides. On the other hand DAVIDSON and WINTER have shown that both 6-MP sensitive and resistant cells con-... 

The details of cell preparations and the assay of the tetrahydrofolate formylase are described elsewhere (12). 

For determination of the tetrahydrofolate formylase activity sodium formate and tetrahydrofolate ere incubated with ATP, ATP-regenerating system and Mg ions in TRIS-puffer pH 7.4 60 minutes at 37 C. By stopping the reaction with perchloric acid all NlO-formyItetrahydrofolic acid is converted to anhydrocitrovorum factor (ACF) which was determined by measuring the absorption at 355 m/u in a Zeiss Spectrophotometer (e=22.000). 

For comparising the activity of the tetrahydrofolate formylase with the netto de novo synthesis of purine nucleotides incubations were performed with 14C-formate and the components necessary for the enzymic formate activation only and in a parallel experiment with additional KHCO3, glutamine,glycine and ribose-5-phosphate (12). Determinations of the tetrahydrofolate formylase activity and of... 

The tetrahydrofolate formylase was found in normal and leukemic leukocytes, the highest activities in immature blast cells of acute Leukemia (table 1). By determination of the 14C-formate incorporation a measurable netto de novo synthesis of purine nucleotides was only detected in immature leukemic cells. 

In figure 6 the influence of 6-MP and allopurinol on the tetrahydrofolate formylase in leukemic blast cells of 5 patients with acute leukemia is illustrated. 6-MP in a rather high concentration of 1.5 x 10 M has only a small inhibitory effect on this enzyme.By addition of allopurinol the inhibition of the enzyme by 6-MP was markedly increased, whereas allopurinol alone had no effect. This is explained by the fact, that allopurinol, by inhibition of the xanthine oxidase, reduces the inactivation of 6-MP to 6-thiouric acid. 

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