A NEW STRUCTURE-BASED REACTION MECHANISM IS PROPOSED FOR UREASE

S Benini¹, S. Ciurli², W. R Rypniewski¹, K. S Wilson³, S Mangani⁴.

¹ EMBL Hamburg Outstation, c/o DESY Notkestrasse 85 Geb 25a 22603 Hamburg Germany
² Istituto di Chimica Agraria Universitaí di Bologna, Viale Berti Pichat 10 40127 Bologna Italy
³ Chemistry Department University of York, Heslington York YO1 5DD United Kingdom
⁴Dipartimento di Chimica Universitaí di Siena, Pian dei Mantellini 44 53100 Siena Italy

Urease (urea aminohydrolase E.C. 3.5.1.5) is a nickel containing enzyme which catalyses the hydrolysis of urea yielding two molecules of ammonia and one molecule of carbon dioxide at a rate 10¹⁴ times faster than the rate of the uncatalysed reaction (1). Ureases have been isolated from bacteria, fungi and plants (2). Urease from Bacillus pasteurii, a highly ureolytic and alkaliphilic soil bacterium has been purified to homogeneity. The enzyme was shown to be heteropolymeric (Mr(a) = 61.4 kDa; Mr(b) = 14.0 kDa; Mr(g) = 11.1 kDa) with a total molecular weight of ca. 230 kDa (3). The coordination of the Ni2+ ions in B. pasteurii urease, studied using X-ray absorption spectroscopy, was shown to consist of five or six nitrogen/oxygen ligands in an octahedral geometry (4). Urease from Bacillus pasteurii has been crystallised (5) . High quality diffraction data have been collected under cryogenic conditions on both native and phenylphosphorodiamidate (PPD) inhibited enzyme. The structure of b-mercaptoethanol inhibited B. pasteurii urease, showing the unexpected chelating mode of binding of this inhibitor (6), has been used as a starting model for the refinement. After 10 cycles of refinement, using REFMAC and ARP for the solvent building the R-factor is 17% and R-free 21% for the native structure and respectively 16.5% and 19.5% for the inhibited.

In the native structure the difference map shows a novel way of clustering of the water molecules in the active site. In the inhibited structure the difference map clearly shows the way of binding of the hydrolysed product of PPD, diamidophosphate, which is proved to be the active molecule for the inhibition reaction (7) and here proposed to resemble the transition state of urea hydrolysis.

The comparison between the two structure allowed us to postulate a new reaction mechanism based on structural evidences.

(1) Hausinger R. P. (1993) Biochemistry of Nickel Plenum Press N.Y.
(2) Mobley H. L. T., Hausinger R.P., (1989) Microbiol. Rev., 53, 85
(3) Benini S,. Ciurli S., Gessa C., (1996) Soil. Biol. Biochem., 28, 819
(4) Benini S., Ciurli S., Mangani S., Nolting H. F. (1996) Eur. J . Biochem., 239, 61.
(5) Benini S., Ciurli S., W. R. Rypniewski, K. S. Wilson, S. Mangani (1998) Acta Cryst., 1998, D54, 409-412.
(6) Benini S., Ciurli S., W. R. Rypniewski, K. S. Wilson, S. Mangani (1998) J. of Bio-Inorg. Chem. in press.
(7) Andrews R. K., Dexter A., Blakeley L., Zerner B. (1986) J. Amer. Chem. Soc., 108, 7124