Allantoate amidinohydrolase (Allantoicase) from Chlamydomonas reinhardtii: its purification and catalytic and molecular characterization.
An allantoate-degrading enzyme has been purified to electrophoretic homogeneity for the first time from a photosynthetic organism, the unicellular green algae Chlamydomonas reinhardtii. The purification procedure included a differential protein extraction followed by conventional steps such as ammonium sulfate fractionation, gel filtration, anion exchange chromatography, and preparative electrophoresis. Under the routine assay conditions (7 mM allantoate), specific activity for the purified enzyme was 185 U/mg, which rose to 225 U/mg under kinetic considerations (saturating substrate). Therefore, a turnover number of 4.5 x 10(4) min(-1) can be deduced for the 200-kDa protein. The enzyme is a true allantoicase (EC 3.5.3.4) that catalyzes the degradation of allantoate to (-)ureidoglycolate and (+)ureidoglycolate to glyoxylate. The enzyme exhibited hyperbolic kinetic for allantoate and ureidoglycolate with K(m) values of 2 and 0.7 mM, respectively. V(max) of the reaction with allantoate as substrate was nine times higher than that with ureidoglycolate. The native enzyme has a molecular weight of 200 kDa and consists of six identical or similar-sized subunits of 34 kDa each, organized in two trimers of 100 kDa. Each subunit has five cysteine residues, four of which are involved in disulfide bonds, with a total of 12 disulfide bonds in the 200-kDa protein. Allantoate inhibits competitively the reaction with ureidoglycolate as substrate. In addition, buffers and group-specific reagents affect the activity in the same manner irrespective of the substrate used. Those results suggest that both substrates use the same active site. The effect of group-specific reagents suggest that the amino acids histidine, tyrosine, and cysteine are essentials for the allantoicase activity with both substrates.
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Departamento de Bioquímica y Biología Molecular, Universidad de Córdoba, Córdoba, 14071, Spain.
Munoz
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Aguilar
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Pineda
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D000596:Amino Acids / Q000737:chemistry; D000645:Ammonium Sulfate / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D002384:Catalysis; D016825:Chlamydomonas reinhardtii / Q000201:enzymology*; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004492:Edetic Acid / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism; D009584:Nitrogen / Q000378:metabolism; D013696:Temperature; D013997:Time Factors; D014512:Ureohydrolases / Q000737:chemistry* / Q000302:isolation & purification* / Q000378:metabolism
D000596:Amino Acids; D004492:Edetic Acid; D014512:Ureohydrolases; C019658:allantoicase; D008274:Magnesium; D009584:Nitrogen; D000645:Ammonium Sulfate