Histidine and Phenylalanine Ammonia-Lyases

B Langer, M Langer, J Retey (2001) Methylidene-imidazolone (MIO) from histidine and phenylalanine ammonia-lyase, Adv Protein Chem 58 175-214... 

The aromatic amino acid L-phenylalanine (primary metabolite) is directed into the phe-nylpropanoid pathway leading to hydroxy-cinnamic acids, lignin and flavonoids by the activity of L-phenylalanine ammonia-lyase (PAL), which brings about its nonoxidative deamination yielding ammonia and tvans-cinnamic acid (Fig. 1). PAL is one of the most studied plant enzymes, and its crystal structure has recently been solved [2]. PAL is related to the histidine and tyrosine ammonia-lyases of amino acid catabolism. A class of bifunctional PALs found in monocotyle-donous plants and yeast can also deaminate tyrosine [3]. A single His residue is responsible for this switch in substrate preference [3, 4]. All three enzymes share a unique MIO (4-methylidene-imidazole-5-one) prosthetic group at the active site. This is formed auto-catalytically from the tripeptide Ala-Ser-Gly by cyclization and dehydration during a late... 

Applications of ammonia lyases Ammonia lyases catalyze the reversible, regio- and stereoselective addition of ammonia to alkenes. The family of lyases includes aspartate, methyl aspartate, histidine, tyrosine and phenylalanine ammonia lyases. The system consisting of phenylalanine ammonia lyase (PAL, EC 4.3.5.1), cinnamic acid, water and ammonium ions was optimized in terms of pH, temperature, reaction time, concentration and buffer for the synthesis of (2S,3S)- and (25,37 )-[3- H]phenylalanines. 

A specialized amino acid residue that serves as an essesn-tial electrophilic center in several enzymatic reactions, including those catalyzed by L-phenylalanine ammonia lyase (Reaction L-phenylalanine tranx-cinnamate + NH3) and L-histidine ammonia lyase (Reaction L-histi-dine urocanate + NH3). The former facilitates the elimination of ammonia and the pro-S hydrogen of phe-nylanine, and the initial step is nucleophilic attack of... 

Catabolism of histidine in most organisms proceeds via an initial elimination of NH3 to form urocanic acid (Eq. 14-44). The absence of the enzyme L-histidine ammonia-lyase (histidase) causes the genetic disease histidinemia 284/285 A similar reaction is catalyzed by the important plant enzyme L-phenylalanine ammonia-lyase. It eliminates -NH3+ along with the pro-S hydrogen in the (3 position of phenylalanine to form frans-cinnamate (Eq. 14-45). Tyrosine is converted to p-coumarate by the same enzyme. Cinnamate and coumarate are formed in higher plants and are converted into a vast array of derivatives (Box 21-E,... 

A large number of a, 3-didehydro-a-amino acids have been identified as constituents of relatively low molecular weight cyclic compounds from microbial sources. However, the presence of a,p-didehydroalanine in bacterial as well as in mammalian histidine ammonia lyase and in phenylalanine ammonia lyase shows that the occurrence of a,p-didehydro-a-amino acids is not limited to small molecules alone 8 These residues are incorporated in natural sequences by posttranslation modification. a,p-Didehydro-a-amino acids have also been postulated to be precursors in the biosynthesis of several heterocyclic metabolites including penicillin and cephalosporin 9 Other well-known compounds containing ,( -di-dehydro-a-amino acids are nisin 10,11 (a food preservative112 ), subtilin (a broad spectrum antibiotic) 13 and some of the metabolites isolated from Streptomyces strains such as gri-seoviridin 14 ... 

Biochemical Mechanisms of Phenylalanine and Tyrosine Ammonia Lyases Comparison to Histidine Ammonia Lyases... 

Histidine ammonia-lyase is the first enzyme in the degradation pathway of L-histidine and catalyzes the nonoxidative deamination of histidine (12) to form w r-urocanic acid (13) plus ammonia (Equation (3)). Histidine ammonia-lyase is present in several bacteria and in animals. The mechanism for the reaction that is catalyzed by histidine ammonia-lyase is presumed to be similar to that described above for phenylalanine ammonia-lyase (see Scheme 3). 

The main pathway of L-histidine degradation includes formation of urocanic acid and leads to glutamic acid (Fig. 241). Histidine ammonia-lyase (like phenylalanine ammonia-lyase, D 22.2.1) catalyzes the transelimination of the NH2-group and a jff-hydrogen atom. Of minor importance in L-histidine degradation is the formation and further degradation of imidazole pyruvic acid. 

Chemical inhibition of L-phenylalanine ammonia lyase activity may be achieved by the use of typical carbonyl reagents such as sodium borohydride and potassium cyanide. Treatment of the enzyme with tritiated sodium borohydride and subsequent hydrolysis gave alanine in which the majority of the radioactivity was confined to the jj-methyl group . Similarly reaction with potassium cyanide and hydrolysis gave aspartic acid labelled exclusively in the -carboxyl group . These observations led to the proposal that the active site of the enzyme, like that of the related L-histidine ammonia lyase , contains a dehydro-alanine residue... 

Dehydroalanine units have been identified at the active sites of the enzymes histidine ammonia lyase 144,434) and L-phenylalanine ammonia lyase (164), either by reduction with NaB Ht (164, 434), or by addition of nitromethane (144) and subsequent reduction to the amine. Following hydrolysis it was possible to detect tritiated alanine or a,y-diamino-butyric acid respectively. 

Decarboxylases of phenylalanine, tyrosine, and lysine and ammonia lyases of histidine, glutamine, and asparagine are also highly selective. Guilbault et al. (1988) described a potentiometric enzyme sensor for the determination of the artificial sweetener aspartame (L-aspartyl-L-phen-ylalanine methylester) based on L-aspartase (EC 4.3.1.1). The ammonia liberated in the enzyme reaction created a slope of 30 mV/decade for the enzyme-covered ammonia sensitive electrode. The specificity of the sensor was excellent however, the measuring time of 40 min per sample appears not to be acceptable. The measuring time has been decreased to about 20 min by coimmobilizing carboxypeptidase A with L-aspartase (Fatibello-Filho et al., 1988). 

A PNH3 electrode covered with this enzyme is used [123] for the determination of L-phenylalanine with very little interference fiom otho amino acids, notably L-tyrosine. A similar selectivity is obtained with phenylalanine decarboxylase [103] which, when attached to a pC02 electrode, enables the specific determination of phenylalanine. Other ammonia lyases are used te determine amino acids. Methionine lyase [124] and histidine ammonia lyase [12S] produce ammonia which is detected with a PNH3 electrode. 

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