Studies on N-acetylneuraminic acid aldolase

Abstract

N-Acetylneuraminic acid aldolase from Clostridium perfringens was irreversibly inactivated by 1mm-bromopyruvate with a half-life of 4.2min at pH7.2 and 37 degrees C. The rate of inactivation was diminished in the presence of pyruvate but not with N-acetyl-d-mannosamine, indicating that the inhibitor acted at, or close to, the pyruvate-binding site. The apparent K(i) for bromopyruvate, calculated from the variation of half-life with inhibitor concentration, was 0.46mm, compared with a competitive K(i) 3.0mm for pyruvate. Incubation of the enzyme with radioactive bromopyruvate gave a radioactive, enzymically inactive, protein in which the bromopyruvate had alkylated cysteine residues. Incubation of the enzyme with radioactive pyruvate, followed by reduction with sodium borohydride, led to inactivation of the enzyme and binding of the pyruvate to the protein by reduction of a Schiff's base initially formed with the in-amino group of a lysine residue; only one-twentieth as many pyruvyl residues were bound by this method, showing that bromopyruvate is not specific for the active site. After protection of the enzyme active site with pyruvate, treatment with unlabelled bromopyruvate and dialysis, the enzyme retained 72% activity. When this treated enzyme was separately incubated with radioactive bromopyruvate, or radioactive pyruvate followed by sodium borohydride, the ratio of radioactive pyruvyl residues bound by the two methods was 2.3:1. After reduction and hydrolysis of the bromopyruvate-treated enzyme, the only detectable radioactive amino acid derivative was chromatographically and electrophoretically identical with S-(3-lactic acid)-cysteine. The enzyme was fully active in the presence of EDTA and was not stimulated by bivalent metal ions. It was strongly inhibited by silver and mercuric ions. The apparent molecular weight, determined by Sephadex chromatography, was 250000. A mechanism of action is proposed for the enzyme. Bromopyruvate reacts rapidly at pH6.0 with thiol-containing amino acids. Cysteine appears to react anomalously.

Purification and Properties of N-Acetylneuraminate Lyase from Escherichia coli1

“…This is the first report that deals with the purification of E. coli A'-acetylneuraminate lyase to a homogeneous state and its detailed characterization. As A'-acetylneuraminate lyase from C. perfringens has been well characterized (4,5,(8)(9)(10)(11)(12)(13)(14)(15)(16), the enzymatic properties of E. coli A'-acetylneuraminate lyase were compared with those of C. perfringens enzyme.…”

“…The enzyme was also found to be widely distributed in animal tissues such as rat liver and brain (5), pig kidney (6), and bovine kidney (7). Among these sources of the enzyme, bacterial Nacetylneuraminate lyase from C. perfringens has been purified and well characterized by several groups of workers (4,5,(8)(9)(10)(11)(12)(13)(14)(15)(16). Although the occurrence of the enzyme was also indicated in Escherichia coli K 235 (5), no report has appeared dealing with the purification of the enzyme to a homogeneous state and its characterization.…”

Characterization of sialidase from Entamoaeba hystolitica and possible pathogenic role in amebiasis

“…The activity of the enzyme pyruvate sialate lyase (PSL) from lysed E. histolytica cells in acetate buffer was assayed as described by Barnett et al (1971).…”

Characterization and mutagenesis of the recombinant N‐acetylneuraminate lyase from Clostridium perfringens

“…The reaction mechanism of N ‐acetylneuraminate lyase from various sources has been studied by many groups (reviewed in [1]). N ‐Acetylneuraminate lyase belongs to the class I aldolases [14,17,25]. During catalysis a Schiff base is formed between the ε‐amino group of a lysine residue and the keto‐function of the substrate.…”