title: Investigation into the Mode of Phosphate Activation in the 4-Hydroxy-4-Methyl-2-Oxoglutarate/4-Carboxy-4-Hydroxy-2-Oxoadipate Aldolase from Pseudomonas putida F1.
abstract: The 4-hydroxy-4-methyl-2-oxoglutarate (HMG)/4-carboxy-4-hydroxy-2-oxoadipate (CHA) aldolase is the last enzyme of both the gallate and protocatechuate 4,5-cleavage pathways which links aromatic catabolism to central cellular metabolism. The enzyme is a class II, divalent metal dependent, aldolase which is activated in the presence of inorganic phosphate (Pi), increasing its turnover rate >10-fold. This phosphate activation is unique for a class II aldolase. The aldolase pyruvate methyl proton exchange rate, a probe of the general acid half reaction, was increased 300-fold in the presence of 1 mM Pi and the rate enhancement followed saturation kinetics giving rise to a KM of 397 ± 30 μM. Docking studies revealed a potential Pi binding site close to, or overlapping with, the proposed general acid water site. Putative Pi binding residues were substituted by site-directed mutagenesis which resulted in reductions of Pi activation. Significantly, the active site residue Arg-123, known to be critical for the catalytic mechanism of the enzyme, was also implicated in supporting Pi mediated activation.
authors.affiliation: Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario, Canada.
authors.forename: Scott
authors.initials: S
authors.lastname: Mazurkewich
authors.affiliation: Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario, Canada.
authors.forename: Stephen Y K
authors.initials: SY
authors.lastname: Seah
mesh_terms: D000446:Aldehyde-Lyases / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D001665:Binding Sites; D004789:Enzyme Activation / Q000187:drug effects*; D007700:Kinetics; D062105:Molecular Docking Simulation; D016297:Mutagenesis, Site-Directed; D007652:Oxo-Acid-Lyases / Q000235:genetics / Q000378:metabolism*; D010710:Phosphates / Q000494:pharmacology*; D017434:Protein Structure, Tertiary; D016958:Pseudomonas putida / Q000201:enzymology*; D016415:Sequence Alignment
chemical_list: D010710:Phosphates; C470744:4-carboxy-4-hydroxy-2-oxoadipate aldolase; D000446:Aldehyde-Lyases; D007652:Oxo-Acid-Lyases; C021412:4-hydroxy-4-methyl-2-oxoglutarate aldolase