A UNIQUE ACTIVE SITE IN A ROBUST ENZYME. Evelyn Jabri, P. Andrew Karplus. Section of Biochemistry, Molecular and Cell Biology, Cornell University, Ithaca, NY, 14853.

The structure of the nickel metalloenzyme urease from Klebsiella aerogenes was solved at 2.2Å resolution (1). The enzyme is a trimer of three ([[alpha]][[beta]][[gamma]])-units, each consisting of four structural domains. The a-subunit contains the active site in an ([[alpha]][[beta]])₈-barrel domain which is homologous to the Zn-dependent enzymes adenosine deaminase and phosphotriesterase. The two active site nickels are 3.5Å apart and have nonstandard coordination geometry. Ni-1 has an unusual tricoordinate geometry whereas Ni-2 is pentacoordinate. Both ions are coordinated by a carbamylated lysine, Lys^[[alpha]]217, explaining why CO₂ is required for the activation of the apoenzyme. We have analyzed the 2.3Å resolution structure (R=19%) of the apoenzyme, and the 2.5Å resolution structures (R=17.9% and 18%, respectively) of the two catalytically impaired active site mutants, H219A and H320A. The final apoenzyme model lacks the CO₂ modification of the lysine and the two nickel ions. Otherwise, the structure of the apoenzyme is nearly identical to that of the holoenzyme, suggesting a high degree of preorganization which helps explain the tight binding of the nickel ions. The major change in the structure of H219A involves a conformational shift and ordering of the active site loop, and a small shift in the side chain of Asp^[[alpha]]221. This latter movement may contribute to the lower activity of H219A. In the structure of H320A, the catalytic water, primarily a Ni-2 ligand in the holoenzyme, shifts into a bridging position. This result shows that the nickel ligation is rather sensitive to the environment at the active site and provides an alternate explanation for the 10⁵-fold lower activity of H320A. These results also show that urease is robust to the loss of nickel ions and active site mutations. Analysis of the tertiary/quaternary structure suggests that the stability of urease may be due to the burial of an unusually large fraction of its residues.

(1) Jabri, Carr, Hausinger, Karplus (1995) Science 268:998-1004.