Investigation into the mechanism of the Bacillus cereus phosphonoacetaldehyde hydrolase enzyme

The enzyme phosphonoacetaldehyde hydrolase, isolated from Bacillus cereus, catalyzes the dephosphonylation of phosphonoacetaldehyde yielding acetaldehyde and phosphate. We determined that the enzyme was inactivated when it was incubated with substrate or acetaldehyde in the presence of NaBH4. The chemical modification was determined to be specific for a single lysine residue by the use of active site radiolabeling methodologies. Phosphonatase was incubated with [3H]-NaBH4 and phosphonoacetaldehyde, [14C]-acetaldehyde/NaBH4, and with [C2-3H]-phosphonoacetaldehyde/NaBH4 which yielded radiolabeled enzyme. The latter of these experiments yielded the most specifically labeled phosphonatase as determined by RP-HPLC separation of the peptides generated by a tryptic digest of the enzyme. The active site peptide was purified to homogeneity and its primary structure determined. var-epsilon-N-Ethyl-L-lysine was identified as the radiolabeled component of the sequence. Acetonyl phosphonate was able to protect phosphonatase from phosphonoacetaldehyde/NaBH4 induced inactivation which suggests that the lysine is in the active site