Sandbox Reserved 644

This Sandbox is Reserved from 30/08/2012, through 01/02/2013 for use in the course "Proteins and Molecular Mechanisms" taught by Robert B. Rose at the North Carolina State University, Raleigh, NC USA. This reservation includes Sandbox Reserved 636 through Sandbox Reserved 685.

To get started: Click the edit this page tab at the top. Save the page after each step, then edit it again. Click the 3D button (when editing, above the wikitext box) to insert Jmol. show the Scene authoring tools, create a molecular scene, and save it. Copy the green link into the page. Add a description of your scene. Use the buttons above the wikitext box for bold, italics, links, headlines, etc. More help: Help:Editing For more help, look at this link: http://proteopedia.org/w/Help:Getting_Started_in_Proteopedia Ornithine TranscarbamoylaseOrnithine Transcarbamoylase IntroductionIntroduction (OTC) is an enzyme that catalyzes the reaction between carbamoyl phosphate and ornithine to form citrulline and phosphate, and this occurs during the second step of the urea cycle. In plants and microbes, OTC is involved in arginine biosynthesis, but in mammals it is located in the mitochondria and is part of the urea cycle. OTC is often associated with Ornithine transcarbamoylase deficiency (OTCD). OTCD is a common urea cycle disorder, and it is a genetic disorder which results in a mutated and ineffective form of the enzyme OTC. The gene is located on the short arm of chromosome X (Xp21.1). The gene is located in the Watson (plus) strand and is 68,968 bases in length. The encoded protein is 354 amino acids long with a predicted molecular weight of 39.935 kiloDaltons. The protein is located in the mitochondrial matrix. StructureStructure OTC is a trimer. The monomer unit has a CP-binding domain and an amino acid-binding domain. Each of the two discrete substrate-binding domains (SBDs) have an α/β topology with a central β-pleated sheet embedded in flanking α-helices. The are located at the interface between the protein monomers.The crystal structure of human ornithine transcarbamylase (OTCase) complexed with carbamoyl phosphate (CP) and L-norvaline (NOR) has been determined to 1.9-A resolution. There are significant differences in the interactions of CP with the protein, compared with the interactions of the CP moiety of the bisubstrate analogue N-(phosphonoacetyl)-L-ornithine (PALO). The carbonyl plane of CP rotates about 60 degrees compared with the equivalent plane in PALO complexed with OTCase. This positions the side chain of NOR optimally to interact with the carbonyl carbon of CP. The mixed-anhydride oxygen of CP, which is analogous to the methylene group in PALO, interacts with the guanidinium group of Arg-92; the primary carbamoyl nitrogen interacts with the main-chain carbonyl oxygens of Cys-303 and Leu-304, the side chain carbonyl oxygen of Gln-171, and the side chain of Arg-330. The residues that interact with NOR are similar to the residues that interact with the ornithine (ORN) moiety of PALO. The side chain of NOR is well defined and close to the side chain of Cys-303 with the side chains of Leu-163, Leu-200, Met-268, and Pro-305 forming a hydrophobic wall. C-delta of NOR is close to the carbonyl oxygen of Leu-304 (3.56 A), S-gamma atom of Cys-303 (4.19 A), and carbonyl carbon of CP (3.28 A). Even though the N-epsilon atom of ornithine is absent in this structure, the side chain of NOR is positioned to enable the N-epsilon of ornithine to donate a hydrogen to the S-gamma atom of Cys-303 along the reaction pathway. Binding of CP and NOR promotes domain closure to the same degree as PALO, and the active site structure of CP-NOR-enzyme complex is similar to that of the PALO-enzyme complex. The structures of the active sites in the complexes of aspartate transcarbamylase (ATCase) with various substrates or inhibitors are similar to this OTCase structure, consistent with their common evolutionary origin. MechanismMechanism results have proved that the mechanism for the formation of argininosuccinate consists of at least two distinct chemical steps with the formation of citrulline adenylate as a reactive intermediate. Argininosuccinate synthetase catalyzes the reversible conversion of citrulline, aspartate, and ATP to argininosuccinate, AMP, and inorganic pyrophosphate. Step 1, activated citrulline-adenylate is formed, releasing inorganic pyrophosphate. Step 2, nucleophilic attack by aspartate amino group forms argininosuccinate and releases AMP (http://www.jbc.org/content/277/15/13074.full#F1). ImplicationsImplications Arginino Succinate Deficiency The argininosuccinic acid synthetase (AS) gene is located on chromosome 9q34.1 spanning 63 kb and composed of 16 exons encoding a protein of 412 amino acids. The functional enzyme exists as a homotetramer. Surprisingly, there are at least 14 AS pseudogenes found on various chromosomes, including two pseudogenes on chromosome 9 but distant from the location of the active AS gene. There are at least 22 known mutations in the AS gene that result in argininosuccinate synthetase deficiency (ASD). Mutations include missense, nonsense and exon deletions. The frequency of ASD is approximately 1 per 57,000 live births. ASD is, like the other neonatal onset forms of UCDs, most severe when presenting in newborn infants. As with each of the four neonatal onset UCDs, ASD is characterized by the accumulation of ammonia and glutamine with clinical manifestations appearing in full-term infants with no prior obstetric risk factors. The classic symptoms appear between 24hrs and 48hrs after birth (but not prior to 24hrs) and include convulsions, hyperventilation, ataxia, hypothermia, lethargy, vomiting and poor feeding. If left untreated the hyperammonemia with result in coma and death. The severe effects of hyperammonemia are described in the Nitrogen Metabolism page. Even though sepsis is a rare event in a normal term infant with no prior obstetric complications, this disorder is misdiagnosed in almost half of neonatal UCD cases. Initial laboratory findings will include respiratory alkalosis which is the earliest objective indication of encephalopathy. The encephalopathy will progress to the point where mechanical ventilation is required. Another routine laboratory finding is reduced serum (blood) urea nitrogen (BUN) which may be as low as 1mg/dl (normal for newborns is 3–12mg/dl). If plasma ammonia levels are not measured the infants' death will be attributed to sepsis, intracranial hemorrhage, or some other disorder that would normally be associated with a pre-term delivery. ASD patients are treated in much the same ways as for other neonatal UCDs in that protein intake must me highly regulated and the hyperammonemia must be controlled. Hemodialysis is the only effective means to rapidly lower serum ammonia levels in these patients. Acute episodes of hyperammonemia can be treated with intravenous administration of Ammunol® and with oral Buphenyl® for chronic adjunctive therapy of hyperammonemia. Additionally, ASD is treated with oral arginine. The utility of arginine therapy stems from the conversion, ultimately, to citrulline by other enzymes of the urea cycle. The arginine is cleaved to urea and ornithine by the action of arginase. Ornithine and carbamoyl phosphate are condensed to citrulline by the action of ornithine transcarbamoylase (OTC). The citulline is then excreted in the urine. Unlike the utility of oral arginine therapy in the treatment of argininosuccinate lyase deficiency (ALD), which leads the excretion of 2 moles of waste nitrogen as argininosuccinate, citrulline only contains 1 mole of waste nitrogen and excretion of citrulline in the urine is not very efficient. Therefore, it is necessary to include sodium phenylbutyrate (or Buphenyl®) in the treatment regimen. (http://themedicalbiochemistrypage.org/as-deficiency.php)