Structure and Mechanism
Human OTC, Pseudomonas aeruginosa OTC, Bacillus subtilis aspartate transcarbamoylase (ATC), and E. coli ATC are all very similar structurally with each polypeptide chain folded into two structural domains, a CP binding domain and an ornithine or aspartate domain.  E. coli has been studied most extensively, and the mechanisms of all function similarly. (4) Human OTC is a homotrimeric protein (Figure 3) with 322 amino acid residues in each polypeptide chain.  The “pre-protein” has a 32 amino acid leader peptide at the N-terminus that is required for directing the protein into the mitochondria, where it is cleaved.(5)  The crystal structure of human OTC complexed with the bisubstrate analog N-phosphonacetyl-L-ornithine (PALO) is shown(see Homepage). The overall topology of each subunit is  alpha/beta, with 14 alpha-helices and 9 beta-sheets. Each subunit is composed of two domains: a CP binding or polar domain and an ORN binding or equatorial domain. The extended C terminus, found only in mammalian OTCs, is part of the CP domain and adjacent to helix H1. Helices H5 and H11 connect the two domains with a hydrophilic cluster consisting of Lys-64, Lys-68, Glu-181, Glu-328, and Asn-329 holding H1, H5, and H11 together.(see Figure 2) (6)

Fig. 2.   Ribbon diagram of human OTCase liganded with  the bisubstrate analog PALO. Alpha-Helices are shown in red, beta-strands in blue, and random coils in yellow. The C-terminal extension (residues 345-354), near helix H1, is shown in green. Helices H11 and H5 are inter-domain helices that link the CP and ORN domains. (6)

 The CP binding domains are located in the interior of the protein, and the binding domains for the ornithine are external. The interface between the subunits is formed primarily by residues from the CP binding domains of adjacent subunits. (6) The sequence of the C-terminal extension, found only in mammals, is homologous to an interhelical loop found in several membrane proteins, including mitochondrial transport proteins, which suggests a possible mode of interaction with the inner mitochondrial membrane. (7)

Fig. 3.   Ribbon diagram of the human OTCase catalytic trimer. The identical subunits are colored pink, blue, and yellow. The bisubstrate analog PALO, shown in red, interacts with residues from two adjacent subunits. Helices H1, H5, and H11 are shown in brighter colors. The C-terminal extensions, shown in green, are  exposed on the convex face of the enzyme. (6)

MECHANISM
OTC undergoes ordered, sequential displacement.  Fist the CP binds in a deep pocket, then the Orn closes up the active site above the CP. (8) Next, the products are formed and released.  The mechanism of forming the citrulline and phosphorus products is as follows.  The sidechain of Cys303 is positioned so as to be able to interact with the gamma-amino group of L-ornithine which attacks the carbonyl C of carbamoyl phosphate in the enzyme-catalyzed reaction. This sulfhydryl group forms a charge relay system with Asp263 and the alpha-amino group of L-ornithine. (7) (See figure 4)


 

Fig 4. The reaction mechanism of OTC. The side chain amino group of Orn undergoes a nucleophilic attack on the carbonyl carbon of CP to form a tetrahedral transition state. Charge rearrangement releases Cit and Pi. (1)

The active site of OTC has been observed using the crystal structure of OTC complexed with thebisubstrate analog PALO. (6)  When compared to the well documented and very similar active site activity  of E. coli ATC, OTC’s active site chemistry has been proposed. (12) PALO mimics the bound CP and Orn, so the amino acid residues that interact with PALO in the acive site are the same residues that interact with the normal substrates. Figure 5 shows the interaction of PALO in the active site of OTC.  Ser-55 through Thr-58, Arg-106, Arg-319, and Gln-82 from the neighboring subunit stabilize the negative charge of the phosphonate of PALO, so they must interact with the phosphate on CP.  The part of PALO that mimics Orn is hydrogen bonded to the main chain carbonyl oxygen of Leu-274, to the nitrogens of Arg-106 and Arg-319, and interacts with Ser-235 and Met-236.  Lys-53, Ser-55, Arg-57, Thr-58, Glu-87, Arg-106, His-133, Gln-136, Asn-167, Asp-231, Ser-235, Met-236, Leu-274, and Arg-319 all interact with the bound substrate analog also. (4) Some scientists were able to bind CP alone to human OTC and found specifically which amino acids in the active site were involved with its binding.  Ser-90,  hr-91, Arg-92, Thr-93, and Arg-141 from one subunit and His-119 from the adjacent subunit are involved in binding the phosphate group of CP.   Gln-191, Cys-303, and Arg-220 are involved in the interaction with the primary nitrogen of CP.  Thr-93, Arg-141, and, His-168 are involved in binding the carbonyl oxygen of CP.  (8) The available crystal structures clearly indicate that the Asp-Xaa-Xaa-Xaa-Ser-Met-Gly sequence in human OTC from the flexible SMG loop is the main binding site for ORN in OTC. (8) (see Figure 6)

Figure 6. Ribbon representation of the OTCase trimer. The three monomers are coloured blue, red and yellow.  Note the SMG loop. (3)