title: Purification, characterization, and in vivo reconstitution of Klebsiella aerogenes urease apoenzyme.
abstract: Urease was purified from recombinant Klebsiella aerogenes which was grown in the absence of nickel. The protein was inactive and contained no transition metals, yet it possessed the same heteropolymeric structure as native enzyme, demonstrating that Ni is not required for intersubunit association. Ni did, however, substantially increase the stability of the intact metalloprotein (Tm = 79 degrees C) compared with apoenzyme (Tm = 62 degrees C), as revealed by differential scanning calorimetric analysis. An increased number of histidine residues were accessible to diethyl pyrocarbonate in apourease compared with holoenzyme, consistent with possible Ni ligation by histidinyl residues. Addition of Ni to purified apourease did not yield active enzyme; however, urease apoenzyme was very slowly activated in vivo by addition of Ni ions to Ni-free cell cultures, even after treatment of the cells with spectinomycin to inhibit protein synthesis. In contrast, sonicated cells and cells treated with dinitrophenol or dicyclohexylcarbodiimide were incapable of activating apourease. These results indicate that apourease activation is an energy-dependent process that is destroyed by cell disruption.
authors: Department of Microbiology, Michigan State University, East Lansing 48824-1101.
authors: M H
authors: MH
authors: Lee
authors: S B
authors: SB
authors: Mulrooney
authors: R P
authors: RP
authors: Hausinger
mesh_terms: D001051:Apoenzymes / Q000302:isolation & purification* / Q000378:metabolism; D001059:Apoproteins / Q000302:isolation & purification*; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D007700:Kinetics; D007711:Klebsiella pneumoniae / Q000201:enzymology*; D000198:Spectinomycin / Q000494:pharmacology; D013816:Thermodynamics; D014510:Urease / Q000302:isolation & purification* / Q000378:metabolism
chemical_list: D001051:Apoenzymes; D001059:Apoproteins; D000198:Spectinomycin; D014510:Urease