Question

Urease is a metalloenzyme; this means it has a metal cofactor. Go to the pdb (protein data bank) and look up the structure 1E9Z. This is the crystal structure of urease from Helicobactor pylori, a common gut bacteria linked to ulcers and stomach cancer.

c. (4)Describe the 3° structure d. (4) Describe the 4° structure e. (4)Describe the cofactor, i.e what metal and how is it attached to the protein? f. (4)Sketch the active site (the metal site)

Answer 1

c.The chains contact with each other to form hydrogen bonds and disulfide bridges.

d. Most bacterial ureases are heteropolymeric enzymes that possess a native Mr of 200,000 to 250,000 (e.g., the proteins recently isolated from L. reuteri , Streptococcus mitior, L. fermentum [306], two filamentous cyanobacteria , and other microorganisms; however, exceptions to this general rule are known (e.g., the heteropolymeric proteins from S. xylosus, S. saprophyticus, M. morganii, and several other species are larger). In particular, the H. pylori enzyme consistently has been reported to be significantly larger, with an Mr of 380,000 to 600,000. For comparison, jack bean urease is known by equilibrium ultracentrifugation studies to have an Mr of ;590,000 and to be composed of a homohexamer of its single subunit. In the presence of glycol, glycerol, and 1,2-propanediol, the native plant enzyme readily dissociates into half-units that retain activity. The stoichiometry of the subunits in bacterial ureases has been the subject of considerable investigation. Separation of individual subunits by denaturing polyacrylamide gel electrophoresis and integration of the staining intensities for the multiple bands reveal near-integer values for the subunit stoichiometries, but these ratios vary for the enzymes from different sources.

e. Urease, the first enzyme to be crystallized, contains a dinuclear nickel metallocenter that catalyzes the decomposition of urea to produce ammonia, a reaction of great agricultural and medical importance. Several mechanisms of urease catalysis have been proposed on the basis of enzyme crystal structures, model complexes, and computational efforts, but the precise steps in catalysis and the requirement of nickel versus other metals remain unclear. Purified bacterial urease is partially activated via incubation with carbon dioxide plus nickel ions; however, in vitro activation also has been achieved with manganese and cobalt. In vivo activation of most ureases requires accessory proteins that function as nickel metallochaperones and GTP-dependent molecular chaperones or play other roles in the maturation process. In addition, some microorganisms control their levels of urease by metal ion-dependent regulatory mechanisms.