Site-directed mutagenesis of Klebsiella aerogenes urease: identification of histidine residues that appear to function in nickel ligation, substrate binding, and catalysis.
Comparison of six urease sequences revealed the presence of 10 conserved histidine residues (H96 in the gamma subunit, H39 and H41 in beta, and H134, H136, H219, H246, H312, H320, and H321 in the alpha subunit of the Klebsiella aerogenes enzyme). Each of these residues in K. aerogenes urease was substituted with alanine by site-directed mutagenesis, and the mutant proteins were purified and characterized in order to identify essential histidine residues and assign their roles. The gamma H96A, beta H39A, beta H41A, alpha H312A, and alpha H321A mutant proteins possess activities and nickel contents similar to wild-type enzyme, suggesting that these residues are not essential for substrate binding, catalysis, or metal binding. In contrast, the alpha H134A, alpha H136A, and alpha H246A proteins exhibit no detectable activity and possess 53%, 6%, and 21% of the nickel content of wild-type enzyme. These results are consistent with alpha H134, alpha H136, and alpha H246 functioning as nickel ligands. The alpha H219A protein is active and has nickel (approximately 1.9% and approximately 80%, respectively, when compared to wild-type protein) but exhibits a very high Km value (1,100 +/- 40 mM compared to 2.3 +/- 0.2 mM for the wild-type enzyme). These results are compatible with alpha H219 having some role in facilitating substrate binding. Finally, the alpha H320A protein (Km = 8.3 +/- 0.2 mM) only displays approximately 0.003% of the wild-type enzyme activity, despite having a normal nickel content.(ABSTRACT TRUNCATED AT 250 WORDS)
Park
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Department of Microbiology, Michigan State University, East Lansing 48824-1101.
Hausinger
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D001483:Base Sequence; D001665:Binding Sites; D002384:Catalysis; D004269:DNA, Bacterial / Q000235:genetics; D004047:Diethyl Pyrocarbonate / Q000494:pharmacology; D004795:Enzyme Stability; D006639:Histidine / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D007460:Iodoacetamide / Q000494:pharmacology; D007700:Kinetics; D007711:Klebsiella pneumoniae / Q000201:enzymology* / Q000235:genetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009532:Nickel / Q000378:metabolism; D014510:Urease / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism
D004269:DNA, Bacterial; D006639:Histidine; D009532:Nickel; D014510:Urease; D004047:Diethyl Pyrocarbonate; D007460:Iodoacetamide