Revisiting the Roles of Catalytic Residues in Human Ornithine Transcarbamylase

Abstract

Human ornithine transcarbamylase (hOTC) is a mitochondrial transferase protein involved in the urea cycle and is crucial for the conversion of toxic ammonia to urea. Structural analysis coupled with kinetic studies of Escherichia coli, rat, bovine, and other transferase proteins has identified residues that play key roles in substrate recognition and conformational changes but has not provided direct evidence for all of the active residues involved in OTC function. Here, computational methods were used to predict the likely active residues of hOTC; the function of these residues was then probed with site-directed mutagenesis and biochemical characterization. This process identified previously reported active residues, as well as distal residues that contribute to activity. Mutation of active site residue D263 resulted in a substantial loss of activity without a decrease in protein stability, suggesting a key catalytic role for this residue. Mutation of predicted second-layer residues H302, K307, and E310 resulted in significant decreases in enzymatic activity relative to that of wild-type (WT) hOTC with respect to l-ornithine. The mutation of fourth-layer residue H107 to produce the hOTC H107N variant resulted in a 66-fold decrease in catalytic efficiency relative to that of WT hOTC with respect to carbamoyl phosphate and a substantial loss of thermal stability. Further investigation identified H107 and to a lesser extent E98Q as key residues involved in maintaining the hOTC quaternary structure. This work biochemically demonstrates the importance of D263 in hOTC catalytic activity and shows that residues remote from the active site also play key roles in activity.

Figure 1

Reaction catalyzed by OTC. The side chain amino group from l-ornithine (blue) performs a nucleophilic attack on the carbonyl carbon atom of carbamoyl phosphate (pink). Charge rearrangement releases the phosphate and forms l-citrulline. l-Ornithine is shown as deprotonated on the basis of current understanding of OTC’s catalytic mechanism.

Figure 2

Crystal structure of hOTC1C9Y displaying the quaternary structure, active site residues, and residues tested. Each monomer subunit is highlighted in a different color. NVA and CP, colored by atom with nitrogen in blue, oxygen in red, phosphate in orange, and carbon in cyan, highlight the residues tested here and their distance from the active site. ORN binding residues 263–267 and 302–305 are colored green. CP binding residues 90–94 and 168–171 are colored yellow. First-shell residues Q171, D263, and C303 are colored pink. Second-shell residues H302, K307, and E310 are colored tan. Third-shell residue E98 and fourth-shell residue H107 are colored brown and dark blue, respectively. Finally, fifth-shell residue A152 is colored orange. Prime and double-prime designations indicate blue and tan subunits, respectively.

Figure 3

Melting temperature (T_m) of WT hOTC and its variants. T_m values are indicated below the graph.

Figure 4

Semilog and Kratky plots for WT hOTC and variants H107N and H302L. (A) Analysis of the Kratky plot showed that the WT protein is folded. (B) The Kratky plot for hOTC variant H107N shows that the protein is folded and the plot lacks the two peaks expected for hOTC. (C) hOTC H302L is folded and the Kratky plot is similar to that of WT hOTC with no indication of a substantial change in protein flexibility. The curve for the apo condition is colored black; the curve for the enzyme with CP is colored red.

Figure 5

Reconstructed envelopes for apo WT hOTC and WT hOTC with CP.

Figure 6

Reconstructed envelopes for apo hOTC H107N and hOTC H107N with CP.

Figure 7

Reconstructed envelopes for apo hOTC H302L and hOTC H302L with CP.

Figure 8

Size exclusion chromatogram comparing WT hOTC and its H107N and E98Q variants, illustrating the difference in the elution profiles and calculated molecular weights of the proteins.

Figure 9

(A) Positions of D263 and C303 relative to each other and to the substrates ORN and CP. (B) Relative position of first-shell residue Q171 in the 168–171 motif implicated in binding of the carbamoyl group of CP. (C) Hydrogen bond between H302 and T262 shown by dotted yellow lines. The 302–305 loop is colored cyan, and the SMG loop purple. (D) Yellow dots show the K307 and E310 hydrogen bonds. The 302–305 loop is colored cyan, and the SMG loop purple.

Figure 10

(A) Trimer center of WT hOTC showing interactions that are present between E98 and H107 in the subunits of the protein. (B) Trimer core of variant H107N, where the interactions between N107 and E98 in the subunits of the protein are weakened. Each subunit is shown in a different color (cyan, purple, and yellow). Prime and double-prime designations indicate the purple and yellow subunits, respectively.