Serine Phosphoric Acid From Diisopropylphosphoryl Chymotrypsin

Mechanisms of Catalysis of Nucleophilic Reactions of Carboxylic Acid Derivatives.

“…Treatment of chymotrypsin with radioactive DFP or Sarin and degradation of the labelled protein gives a series of phosphopeptides from which amino acid sequences have been obtained. The amino acid sequence at the active site of chymotrypsin (334,336,385), trypsin (130,131,132,307,333), liver aliesterase (224a), pseudocholinesterase, thrombin (174), and phosphoglucomutase where GLY is glycine, ASP is aspartic acid, GLU is glutamic acid, SER is serine and ALA is alanine, and where ASP or GLU and GLY or ALA are alternatives in the sequence. The common sequence of amino acids around serine for many hydrolytic enzymes is rather surprising.…”

Alkylphosphorylation of Stem Bromelain by Diisopropylphosphorofluoridate without Inhibition of Proteinase Activity*

“…The inhibition was also found to occur with other proteolytic enzymes like thrombin, elastase, and subtilisin (Hartley, 1960). The inhibition by DFP has such characteristic features common to all these proteinases that (1) DFP rapidly alkylphosphorylates the enzyme protein, (2) the introduction of a single diisopropylphosphoryl (DIP)1 group per molecule of enzyme protein results in a total loss of activity, and (3) the site of alkylphosphorylation of the enzyme protein is always one of the serine hydroxyls as evidenced by isolation of either O-phosphorylserine from an acid hydrolysate of DIP enzyme (Schaffer et al, 1953) or a peptide containing an O-DIP-seryl residue from an phorylation also occurs even when the essential SH group of the enzyme has been blocked by mercuric ion. The phosphorus incorporated is so firmly bound to the enzyme protein that it can neither be removed by gel filtration nor by repeated ammonium sulfate precipitation and dialysis, indicating a true alkylphosphorylation.…”

Biochemistry

“…Attempts have been made to identify the site of reaction of DFP in chymotrypsin or acetylcholinesterase by acid or enzymic hydrolysis of the inhibited enzyme containing 32P tracer. 83 In this way O-phosphorylserine and O-phosphorylserylglycine have been obtained as hydrolysis products. The authors point out that these products may have arisen by transfer of a labile phosphoryl residue from its original site in the active centre.…”