Protein carbamoylation

Only a few important representatives of the non-proteinogenic amino acids are mentioned here. The basic amino acid ornithine is an analogue of lysine with a shortened side chain. Transfer of a carbamoyl residue to ornithine yields citrulline. Both of these amino acids are intermediates in the urea cycle (see p.l82). Dopa (an acronym of 3,4-dihydroxy-phenylalanine) is synthesized by hydroxyla-tion of tyrosine. It is an intermediate in the biosynthesis of catecholamines (see p.352) and of melanin. It is in clinical use in the treatment of Parkinson s disease. Selenocys-teine, a cysteine analogue, occurs as a component of a few proteins—e.g., in the enzyme glutathione peroxidase (see p.284). 

At least two gene products, the putative carbamoylsynthase TobL and the carbamoyltransferase TobZ are needed for the carbamoylation found in the NEBs. The respective enzymes are unique among the AGA gene clusters analyzed so far. Because NEBs are mainly found as a complex of NEB-4 and NEB-5, the TobZ protein must recognize both KAN-B and TOB as substrates. Therefore the carbamoylation must be the final step in the NEB biosynthesis, as illustrated in Figure 2.16b. 

The prototypical allosteric regulatory protein isolated from Escherichia coli (molecular weight 310,000). The enzyme [EC 2.1.3.2], also known as aspartate transcarba-mylase and carbamylaspartotranskinase, catalyzes the first committed step i.e., the transfer of a carbamoyl... 

Fig. 38. The zinc-thiolate cluster of the regulatory subunit of aspartate carbamoyl-transferase (Honzatko et al 1982). Coordinates are from the Brookhaven Protein Data Bank.

Enzyme activity on a D (non-natural) configuration, non-protein cyclic amino acid derivative appears unlikely. However the D-hydantoinase reaction is very similar to the dihydroxypyrimidase present in pyrimidine metabolism. The original hydantoinase used was obtained from calf liver, but subsequently many active microorganisms were found, particularly a strain of B. brevis. The resulting D-A-carbamoyl amino acid can then be converted into product by treatment with nitrous acid (Figure 4.11). 

The down-regulated proteins in HCC tissues have been identified. Park et al. identified aldehyde dehydrogenase 2 (25) and ferritin light chain (32). Kim et al. identified HSP 27, cathepsin D, and others (26). Lim et al. identified cytochrome B5, liver car-boxyesterase, and others (27). Li et al. identified SOD 1, aldolase B, and others (28). Fujii et al. identified galectin-1 (29). Kim et al. identified argininosuccinate synthase, carbamoyl-phosphate s mthase, and others (31). Table 1 shows the summary of the proteins whose expression was different between HCC cancer tissues and non-cancerous tissues. 

Table 2.1.9 Changes of blood amino acids in various primary inherited defects and as a result of secondary changes. ASA Argininosuccinic acid, CPS carbamoyl phosphate synthase, LPI Lysinuric protein intolerance, MAD multiple acyl-CoA dehydrogenation, MSUD maple syrup urine disease, NAGS N-acetylglutamate synthase, NKH nonketotic hyperglycinemia, NTBC 2-(2-nitro-4-3 trifluoro-methylbenzoyl)-1,3-cyclohexanedione, OCT Ornithine carbamoyltransferase,...

Marina, A. Alzari, P.M. Bravo, J. Uriarte, M. Barcelona, B. Fita, L Rubio, V. Carbamate kinase new structural machinery for making carbamoyl phosphate, the common precursor of pyrimidines and arginine. Protein Sci., 8, 934-940 (1999)... 

These changes in demand for urea cycle activity are met over the long term by regulation of the rates of synthesis of the four urea cycle enzymes and carbamoyl phosphate synthetase I in the liver. All five enzymes are synthesized at higher rates in starving animals and in animals on veiy-high-protein diets than in well-fed animals eating primarily carbohydrates and fats. Animals on protein-free diets produce lower levels of urea cycle enzymes. 

Formation of citrulline Ornithine and citrulline are basic amino acids that participate in the urea cycle. [Note They are not incorporated into cellular proteins, because there are no codons for these amino acids (see p. 429).] Ornithine is regenerated with each turn of the urea cycle, much in the same way that oxaloacetate is regenerated by the reactions of the citric acid cycle (see p 109). The release of the high-energy phosphateof carbamoyl phosphate as inorganic phosphate drives the reaction in the forward direction. The reaction product, citrulline, is trans ported to the cytosol. 

The binding of PALA to the R conformation of aspartate carbamoyl transferase. Arg 105 and His 134 are provided by one domain of a c subunit, and Arg 167 and Arg 229 by the other domain. Ser 80 and Lys 84 are part of a loop of protein from a different c subunit. The PALA is indicated by red. The wavy green lines indicate polypeptide backbone structure. 

The complete urea cycle as it occurs in the mammalian liver requires five enzymes Argininosuccinate synthase, arginase, and argininosuccinate lyase (which function in the cytosol), and ornithine transcarbamoylase, and carbamoyl phosphate synthase (which function in the mitochondria). Additional specific transport proteins are required for the mitochondrial uptake of L-ornithine, NH3, and HC03 and for the release of L-citrulline. 

Low activities of orotidine phosphate decarboxylase and (usually) orotate phosphoribosyltransferase are associated with a genetic disease in children that is characterized by abnormal growth, megaloblastic anemia, and the excretion of large amounts of orotate. When affected children are fed a pyrimidine nucleoside, usually uridine, the anemia decreases and the excretion of orotate diminishes. A likely explanation for the improvement is that the ingested uridine is phosphorylated to UMP, which is then converted to other pyrimidine nucleotides so that nucleic acid and protein synthesis can resume. In addition, the increased intracellular concentrations of pyrimidine nucleotides inhibit carbamoyl phosphate synthase, the first enzyme in the. naibwav of aro-tate synthesis. 

The entry of activated ammonia into the urea cycle occurs by the ornithine transcarbamoylase reaction where the carbamoyl group is transferred to the side chain amino group of the non-protein amino acid, ornithine. Ornithine has five carbons its carbon chain therefore has the same length as that of arginine. The product of the ornithine transcarbamoylase reaction is the amino acid citrulline. 

In other studies on the domain structure of oligomeric proteins, limited proteolysis, deletions, and site-directed mutagenesis have also successfully provided information about subunit contact regions. Functional domains containing subunit interaction sites of cAMP-dependent protein kinase22 and carbamoyl phosphate synthetase23 have been revealed by such experiments. 

---