Histidine nitrogen removal

Not included in this simplified description of the active site is the important role played by another amino acid residue. The basicity of the histidine nitrogen is increased because of the proximity of a neighbouring aspartate residue. This facilitates removal of a proton from the active site serine. The relationship of these three residues... 

The derivatives are easily prepared by treating amino sugars with 2,4-dinitrofluorobenzene in the presence of sodium carbonate and have been used for characterization purposes. Recently, this group has also been used to protect the ring nitrogen atom in histidine [195] removal was brought about by thiolysis at pH 8 with aqueous 2-aminoethanol. 

A typical role for the histidine imidazole ring is shown below, in the enzyme mechanism for a general base-catalysed hydrolysis of an ester. The imidazole nitrogen acts as a base to remove a proton from water, generating hydroxide that attacks the carbonyl. Subsequently, the alkoxide leaving group is reprotonated by the imidazolium... 

Serine itself would be insufficiently nucleophilic to attack the ester carbonyl, so the reaction is facilitated by participation of the imidazole ring of histidine. The basic nitrogen in this residue is oriented so that it can remove a proton from the serine hydroxyl, increasing nucleophilicity and allowing attack on the ester carbonyl. This leads to formation of the transient acetylated enzyme, and release of choline. Hydrolysis of the acetylated enzyme utilizes water as nucleophile, but again involves the imidazole ring, and regenerates the free enzyme. 

The removal of iron from such species as transferrin or ferritin by hydroxypyridinones, side-rophores, and other chelators is of considerable relevance to the control of iron levels in the body, and indeed to iron metabolism in a range of life forms. Methods and mechanisms for such removal are referenced in Sections 5.4.5.2,5.4.5.5.2, 5.4.5.6.1, and 5.4.5.6.2 below. Interestingly cyanide, one of the most powerful ligands for iron, appears to prefer to bind to iron-transferrin, at the C-terminal Fe, rather than to remove the iron. This adduct is believed to contain the iron in an octahedral environment of three cyanide ligands mer) and nitrogens from two tyrosine residues and a histidine. ... 

The electrostatic potential calculated for one molecule removed from the crystal lattice in the histidine plane (Figure 19a) shows a very small minimum of potential (-0.18 elk 1) around the nitrogen atom which becomes positive when the calculation is made for a cluster of two hydrogen-bonded molecules [53] (N4—Ns = 2.856 A) (Figure 19b). 

At the active site of beef heart mitochondrial succinate dehydrogenase (EC 1.3.99.1), the FAD is covalently linked by C-8a to nitrogen of a histidine residue (Fig. 2b) [20]. In the catalytic reaction, removal of a proton from C-3 of the succinate may be followed by attack of the 3-carbanion on N-5 of the FAD to form an intermediate adduct, which breaks down with loss of a proton from C-2 of the succinate, giving fumarate and a reduced FAD moiety [21]. This mechanism is not certain, but it is established that the succinate loses two non-equivalent hydrogen atoms by a trans elimination (Fig. 4) [22], In other enzymes, different types of covalent attachment of the FAD are known [23]. 

Nitrogens 1 and 3 in the imidazole ring of histidyl residues in proteins may be alkylated with iodoacetic acid (generally in a much slower reaction than alkylation of cysteinyl residues) to give three carboxy-methyl derivatives 1-carboxymethylhistidine, 3-carboxymethyl-histidine and 1,3-dicarboxymethylhistidine ( 3.4). In general, the 3-carboxymethyl derivative is formed most rapidly. These derivatives are stable to acid hydrolysis under the usual conditions (but excess reagent must be removed) and may be analyzed on the long column of most analyzers as described below. 

The combined ether extract (which may be washed with 0.01 N HCl to remove peptide material) is evaporated to dryness with a stream of nitrogen and 50 /il of 1 N HCl is added. After mixing, the thiazolinone derivative of the NH2 terminal residue is converted to the phenyl-thiohydantoin by incubation at 80°C (temperature block) for 10 min. If Asx or Glx residues are expected, 80°C for 3 min may give better results and for threonine, proline and serine, 50°C for 10 min may be better (Li and Yanofsky 1972). After cooling, the phenylthiohydantoin is extracted into ethyl acetate (100-200 /rl for each of 3 extractions), which is then evaporated with a stream of nitrogen. The phenylthio-hydantoins of arginine, histidine and cysteic acid will usually remain in the aqueous phase and may be recovered by lyophilization and dissolving the residue in methanol. 

The pH dependence of the kinetics of histidine decarboxylase (127) demonstrates that the histidine is zwitterionic when it binds to the enzyme. The extra proton on nitrogen must, of course, be removed before the Schiff base is formed. The carboxylate of Glu-197 at the active site may accept this proton. In turn, this same group may then be responsible for proton donation to the Schiff base following decarboxylation. This is consistent with the occurrence of retention of configuration in the overall replacement of-C02 by -H (128) and with studies of enzymes altered at Glu-197 (129). When Glu-197 is replaced by Asp, the protonation that follows decarboxylation occasionally occurs on the pyruvate side, thus giving rise to decarboxylation-dependent transamination (129). 

---