Active formate from histidine

More recently Brown and Kies (S65) reported the formation from histidine and the excretion in the urine of hydantoin- propionic acid, and also its formation by liver extracts of guinea pig and the rat. The substrate for the oxidation was shown very probably to be imidazolone-propionic acid. No oxidation or formation of hydantoinpropionic acid could be demonstrated if the urocanase activity was first destroyed. The L-hydantoin-5-propionic acid was isolated by chromati raphy, and crystallized. Its identity was unequivocally established. 

Amine build-up in fish muscle usually results from decarboxylation of amino acids in the muscle by enzymes of bacterial origin. This review will present information on the activity of bacterial decarboxylases and the formation of amines in fish. Mechanisms of decarboxylase action and production of bacterial decarboxylases in fish muscle are discussed. Emphasis is placed upon studies dealing with formation of histidine decarboxylase and histamine. Histamine, because of its involvement in Scombroid food poisoning, has been extensively studied with regard to its formation in fish and fishery products. 

The reaction mechanism catalysed by sEH has been recently elucidated from experiments using heavy isotopes, protein, mass spectrometry, site-directed mutagenesis, and has been supported by the recent crystal structure determination at 2.8-A resolution (Fig. 31.28). This two-step reaction mechanism involves a catalytic nucleophile (aspartic acid 333) which can attack the polarized epoxide ring by two tyrosyl residues (tyrosines 381 and 465) leading to the ring opening and the formation of an acyl-enzyme intermediate. The second step corresponds to hydrolysis of this intermediate by a water molecule activated by a histidine 523-aspartic acid 495 pair." ... 

Serine proteases (e.g., trypsin, chymotrypsin, subtilisin) catalyze the hydrolysis of ester or amide substrates through an acyl-enzyme intermediate in which the hydroxyl group of the active serine side chain is acylated by the substrate (Figure 5), The reaction involves proton transfer from serine to the catalytic histidine residue, an attack on the amide carbon of the substrate, and formation of a high-energy tetrahedral intermediate. Subsequently, this intermediate breaks down, the leaving group accepts a proton from histidine and an acyl enzyme is formed which is then hydrolyzed via the reverse route. 

The movement of the proton from histidine to aspartate (as suggested by the charge relay mechanism in serine proteases, cf. Section 4.1) was excluded on the basis of the higher activation energy obtained for this process. Such a proton transfer may be hindered by the electrostatic effect of the environment favoring the formation of the ion pair. 

The lipase (PAL) used in these studies is a hydrolase having the usual catalytic triad composed of aspartate, histidine, and serine [42] (Figure 2.6). Stereoselectivity is determined in the first step, which involves the formation of the oxyanion. Unfortunately, X-ray structural characterization of the (S)- and (J )-selective mutants are not available. However, consideration of the crystal structure of the WT lipase [42] is in itself illuminating. Surprisingly, it turned out that many of the mutants have amino acid exchanges remote from the active site [8,22,40]. 

After the nucleophilic attack by the hydroxyl function of the active serine on the carbonyl group of the lactone, the formation of the acyl-enzyme unmasks a reactive hydroxybenzyl derivative and then the corresponding QM. The cyclic structure of the inhibitor prevents the QM from rapidly diffusing out of the active center. Substitution of a second nucleophile leads to an irreversible inhibition. The second nucleophile was shown to be a histidine residue in a-chymotrypsin28 and in urokinase.39 Thus, the action of a functionalized dihydrocoumarin results in the cross-linking of two of the most important residues of the protease catalytic triad. 

The enzyme catalyzing the formation of retinal 2 by means of central cleavage of P-carotene 1 has been known to exist in many tissues for quite some time. Only recently, however, the active protein was identified in chicken intestinal mucosa (3) following an improvement of a novel isolation and purification protocol and was cloned in Escherichia coli and BHK cells (4,5). Iron was identified as the only metal ion associated with the (overexpressed) protein in a 1 1 stoichiometry and since a chromophore is absent in the protein heme coordination and/or iron complexation by tyrosine can be excluded. The structure of the catalytic center remains to be elucidated by X-ray crystallography but from the information available it was predicted that the active site contains a mononuclear iron complex presumably consisting of histidine residues. This suggestion has been confirmed by... 

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