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

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 (P(i)), 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 P(i) and the rate enhancement followed saturation kinetics giving rise to a K(M) of 397 ± 30 μM. Docking studies revealed a potential P(i) binding site close to, or overlapping with, the proposed general acid water site. Putative P(i) binding residues were substituted by site-directed mutagenesis which resulted in reductions of P(i) activation. Significantly, the active site residue Arg-123, known to be critical for the catalytic mechanism of the enzyme, was also implicated in supporting P(i) mediated activation.