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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.[1]
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.[2]
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.[3]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.<ref>http://www.proteopedia.org/wiki/index.php/1c9y<r/ref>
References:
Argininosuccinate SynthetaseArgininosuccinate Synthetase
StructureStructure
The overall and subunit structure of tAsS is quite similar to that of E. coli AsS, although the precise structure is different between them, probably because the polypeptide chain of tAsS is 47 amino acid residues shorter than that of E. coli AsS. The tAsS is folded into a tetrameric form with a noncrystallographic D2 symmetry, having the shape of a twisted rectangle with an active site at each of the four corners. In the center of the molecule, there are clusters of �-helices which are surrounded by �-sheets. One subunit in the tetramer interacts with the other three subunits and the surface areas of the subunit interfaces are 4,390 for a and b, 1201 for a and d, and 923 Å 2 for a and c subunits. The largest of these areas is found between two subunits of a and b or c and d, indicating that the tetramer may be considered to be an assembly of two dimer units (a dimer of dimers) around a 2-fold axis. All the subunit interfaces are
distant from the active site and not essential for the catalytic action. The C� carbon atoms of the subunit in the native tAsS can be superimposed onto the corresponding ones in tAsS�ATP and tAsS�AMP-PNP�arginine�succinate within r.m.s. deviations of 0.14 and 0.19 Å with the maximum displacement of 0.88 and 0.73 Å, respectively. Thus, tAsS does not significantly change its conformation upon binding of the ATP (AMP-PNP) and substrate analogues. Similarly, E. coli AsS does not change its overall conformation on substrate binding, although the enzyme shows a few localized conformational changes. The C� positions of the native E. coli AsS and the complex one with citrulline and aspartate superimpose within r.m.s. deviations of 0.6 Å. The subunit superposition of tAsS�AMP-PNP�arginine�succinate and E. coli AsS�citrulline�aspartate resulted in 284 equivalenced C� atoms with r.m.s. deviations of 0.54 Å with the maximum displacement of 2.98 Å, indicating that the overall structures of both subunits are essentially the
same. The subunit is divided into a small domain (ATP binding domain, N-terminal to Pro 165), a large domain (Val 166 to Arg 359), and a C terminal arm (Gln 360 to C-terminal). The small domain has a typical �/� structure of an open parallel �-sheet and is folded into the structure similar to that of the ATP pyrophosphate domains of the GMP, NAD�, and asparagine synthetases (3, 5, 7). The five �-strands designated as b3, b2, b1, b4, and b5 (all parallel) form a twisted �-sheet structure as a central core surrounded by two �-helices (H1 and H2) from the convex surface side of the sheet and two �-helices (H4 and H5) from the concave side. The residues from Phe 69 to Ala 91 (�-helix H3 a loop �-helix H4) go through the large hole formed at the center of the Cterminal domain, and the loop reaches the exit of the hole to interact with the other subunit of the dimeric unit. Thus, this region of the N-terminal domain behaves like a part of the Cterminal one. The �-helix H7 covers the �helix H1, and its C-terminal loop goes to the C-terminal domain. The large domain consists of four stranded antiparallel-sheets (b6, b7, b12, and b11), five stranded antiparallel-sheets (b10, b9, b8, b13, and b14), and four �-helices. Two�-sheets of the large domain are connected through the �-helix H8 between the �-strands of b10 and b11 and a succession of three �-helices of H9, H10, and H11 between the �-strands of b12
and b13. The four-stranded �-sheet adjacent to the small domain and �-helices H9, H10, and H11 make a large hole going through the center of the large domain. The long C-terminal arm region reaches the small domains of the other subunits with its Cterminal �-helix interacting with them. When the arm is neglected, the surface areas of the subunit interfaces for a and b, a and d, and a and c subunits are reduced to 2,750, 11, and 654 Å 2, respectively, indicating that the arm is extensively involved in the subunit interactions as the joint for the formation of a dimer or a tetramer.
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