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The crystal structure of human ornithine transcarbamylase (OTCase) complexed with carbamoyl phosphate (CP) and L‐norvaline (NOR) has been determined to 1.9‐Å 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° 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‐δ of NOR is close to the carbonyl oxygen of Leu‐304 (3.56 Å), S‐γ atom of Cys‐303 (4.19 Å), and carbonyl carbon of CP (3.28 Å). Even though the N‐ϵ atom of ornithine is absent in this structure, the side chain of NOR is positioned to enable the N‐ϵ of ornithine to donate a hydrogen to the S‐γ 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. Proteins 2000;39:271–277. © 2000 Wiley‐Liss, Inc.

Tyr 165 in the catalytic subunit of Escherichia coliaspartate transcarbamoylase (ATCase, EC 2.1.3.2) forms an intersubunit hydrogen bond in the T state with Glu 239 in the 240s loop of a second catalytic subunit, which is broken in the T to R transition. Substitution of Tyr 165 by Phe lowers substrate affinity by approximately an order of magnitude and alters the pH profile for enzyme function. We have determined the crystal structure of Y165F at 2.4 Å resolution by molecular replacement, using a wild‐type T state structure as the probe, and refined it to an R value of 25.2%. The Y165F mutation induces a global conformational change that is in the opposite direction to the T to R transition and therefore results in an extreme T state. The two catalytic trimers move closer by ∼0.14 Å and rotate by ∼0.2°, in the opposite direction to the T→R rotation; the two domains of each catalytic chain rotate by ∼2.1°, also in the opposite direction to the T→R transition; and the 240s loop adopts a new conformation. Residues 229 to 236 shift by ∼2.4 Å so that the active site is more open. Residues 237 to 244 rotate by ∼24.1°, altering interactions within the 240s loop and at the C1‐C4 and C1‐R4 interfaces. Arg 167, a key residue in domain closure and interactions with L‐Asp, swings out from the active site to interact with Tyr 197. This crystal structure is consistent with the functional properties of Y165F, expands our knowledge of the conformational repertoire of ATCase, and indicates that the canonical T state does not represent an extreme. Proteins 33:430–443, 1998. © 1998 Wiley‐Liss, Inc.

Tyr 165 in the catalytic subunit of Escherichia coli aspartate transcarbamoylase (ATCase, EC 2.1.3.2) forms an intersubunit hydrogen bond in the T state with Glu 239 in the 240s loop of a second catalytic subunit, which is broken in the T to R transition. Substitution of Tyr 165 by Phe lowers substrate affinity by approximately an order of magnitude and alters the pH profile for enzyme function. We have determined the crystal structure of Y165F at 2.4 Å resolution by molecular replacement, using a wild-type T state structure as the probe, and refined it to an R value of 25.2%. The Y165F mutation induces a global conformational change that is in the opposite direction to the T to R transition and therefore results in an extreme T state. The two catalytic trimers move closer by ~0.14 Å and rotate by ~0.2°, in the opposite direction to the T→R rotation; the two domains of each catalytic chain rotate by ~2.1°, also in the opposite direction to the T→R transition; and the 240s loop adopts a new conformation. Residues 229 to 236 shift by ~2.4 Å so that the active site is more open. Residues 237 to 244 rotate by ~24.1°, altering interactions within the 240s loop and at the C1-C4 and C1-R4 interfaces. Arg 167, a key residue in domain closure and interactions with L-Asp, swings out from the active site to interact with Tyr 197. This crystal structure is consistent with the functional properties of Y165F, expands our knowledge of the conformational repertoire of ATCase, and indicates that the canonical T state does not represent an extreme. Proteins 33:430–443, 1998. © 1998 Wiley-Liss, Inc.