Purification and characterization of allantoate amidohydrolase from Bacillus fastidiosus.
Allantoate amidohydrolase from Bacillus fastidiosus was purified 170-fold to homogeneity as judged by isoelectric focusing and nondenaturing and sodium dodecyl sulfate polyacrylamide gel electrophoresis. The molecular mass was estimated to be 128 kDa. The enzyme appeared to be a homodimer with a subunit molecular mass of 66 kDa. The enzyme has an isoelectric point of 5.6. Allantoate amidohydrolase is a Mn(2+)-dependent enzyme exhibiting a pH optimum around 8.8. Its Km value for allantoate was estimated to be 9 mM. Similar to other microbial allantoate amidohydrolases the enzyme can be reversibly activated and inactivated. No indication for the involvement of arginine, lysine, and cysteine residues in the catalytic action of the enzyme was obtained. Diethylpyrocarbonate strongly inhibited the enzyme activity, indicating the involvement of histidine or tyrosine residues in catalytic action. However, no recovery was obtained by treatment with hydroxylamine as would be expected if such residues were modified. The enzyme could be reversibly denatured by urea, guanidine, and sodium dodecyl sulfate.
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Department of Microbiology and Evolutionary Biology, Faculty of Science, University of Nijmegen, The Netherlands.
de Windt
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van der Drift
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D000481:Allantoin / Q000378:metabolism*; D001407:Bacillus / Q000201:enzymology*; D004047:Diethyl Pyrocarbonate / Q000494:pharmacology; D004789:Enzyme Activation; D019791:Guanidine; D006146:Guanidines / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D019811:Hydroxylamine; D006898:Hydroxylamines / Q000494:pharmacology; D007526:Isoelectric Point; D007700:Kinetics; D011487:Protein Conformation; D011489:Protein Denaturation; D014508:Urea / Q000494:pharmacology; D014512:Ureohydrolases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism
D006146:Guanidines; D006898:Hydroxylamines; D019811:Hydroxylamine; D000481:Allantoin; D014508:Urea; D014512:Ureohydrolases; C019657:allantoate amidohydrolase; D019791:Guanidine; D004047:Diethyl Pyrocarbonate