The reaction of N-acetylneuraminate lyase with chloropyruvate

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Abstract
Chloropyruvate, like bromopyruvate, rapidly inactivates N-acetylneuraminate lyase at pH7.2. At 5°C, 0.5mm-chloropyruvate reacted with the enzyme about ten times as fast as bromopyruvate. In contrast, at pH6.0 and 9°C, chloropyruvate reacted with N-acetylcysteine seven times more slowly than bromopyruvate. A brief (2min) incubation of the enzyme with 1.0mm-chloro[14C]pyruvate gave an inactive enzyme in which 4.5 cysteine residues were alkylated per molecule of enzyme. This corresponded to the number of [14C]pyruvate residues (3.7) bound to the enzyme by borohydride reduction of the [14C]-pyruvate complex, and confirmed the previous suggestion that there is one ‘essential’ cysteine residue per active site. It is suggested that, for this enzyme, chloropyruvate can be selectively used to alkylate the active-site residues, whereas bromopyruvate cannot. The apparent molecular weight of the enzyme prepared by two slightly different methods was approx. 100000 or 250000. The latter value has been used to calculate the number of pyruvate residues bound to the enzyme.