X-ray structure and characterization of carbamate kinase from the human parasite Giardia lamblia

Abstract

Carbamate kinase catalyzes the reversible conversion of carbamoyl phosphate and ADP to ATP and ammonium carbamate, which is hydrolyzed to ammonia and carbonate. The three-dimensional structure of carbamate kinase from the human parasite Giardia lamblia (glCK) has been determined at 3 A resolution. The crystals belonged to the monoclinic space group P2(1), with unit-cell parameters a = 69.77, b = 85.41, c = 102.1 A, beta = 106.8 degrees . The structure was refined to a final R factor of 0.227. The essentiality of glCK together with its absence in humans makes the enzyme an attractive candidate for anti-Giardia drug development. Steady-state kinetic rate constants have been determined. The k(cat) for ATP formation is 319 +/- 9 s(-1). The K(m) values for carbamoyl phosphate and ADP are 85 +/- 6 and 70 +/- 5 microM, respectively. The structure suggests that three invariant lysine residues (Lys131, Lys216 and Lys278) may be involved in the binding of substrates and phosphoryl transfer. The structure of glCK reveals that a glycerol molecule binds in the likely carbamoyl phosphate-binding site.

Figure 1

Overall fold of glCK. A ribbon-diagram representation of the protein dimer is shown. Each subunit is represented in a different color. The glycerol molecules in the active sites are shown in red.

Figure 2

View of the glCK active site. (a) The σ_A-weighted 2F _o − F _c difference Fourier electron-density map in the vicinity of the glycerol molecule (labeled GL) calculated omitting the ligand from the model. Atomic colors are as follows: carbon, yellow; oxygen, red; nitrogen, blue; sulfur, green. (b) The lysine and aspartic acid cluster in the vicinity of the glycerol molecule. Atomic colors are as follows: carbon, gray (glCK) and green (glycerol, labeled GL); oxygen, red; nitrogen, blue.

Figure 3

Stereoscopic view of the glCK active site with modeled Mg²⁺-ADP and carbamoyl phosphate. The Mg²⁺-ADP was modeled by analogy to that observed in the pfCK structure (Ramon-Maiques et al., 2000 ▶). The same color scheme is used for carbon, oxygen and nitrogen as in Fig. 2 ▶(b); P atoms are colored orange.

Figure 4

Superposition of glCK (green) and pfCK (gray) showing the environment of the adenine group of ADP. A tyrosine-carrying loop adopts a more closed conformation in pfCK than in glCK such that the tyrosine residue stacks against the adenine ring. A loop that flanks the other side of the adenine ring is partially disordered in the crystal structure of glCK, which does not contain ADP.