Acylneuraminate pyruvate lyase

Tentative localization of acetyl groups at the 0-4 atoms of sialic acids in native complex carbohydrates is possible with the aid of siali-dases, which are almost, or completely, inactive with these compounds or, after isolation of the sialic acids, by acylneuraminate pyruvate-lyase, which also shows little activity with these substrates (see Section VI). 

That secretion of 2-acetamidoglucal, which is known to be an intermediate in the complex, 2-epimerase reaction, but cannot be isolated under normal conditions, may point to another defect in this enzyme that may be independent of the presence or absence of a feedback-inhibition receptor site for CMP-Neu5Ac has been discussed.233 An excess of 2-acetamidoglucal may be converted spontaneously into GlcNAc and this, enzymically, into ManNAc both compounds are secreted in the urine of the sialuria patient. As a consequence of these reactions, the cellular concentration of ManNAc may increase to a level leading to the synthesis of additional Neu5Ac from this compound by the action of acylneuraminate pyruvate-lyase. All of these reactions, and known or theoretical interactions, were summarized in Fig. 3 of Ref. 233. 

Scheme 4.—Induction and Cooperation of the Sialic acid-specific Enzymes Siali-dase, Acylneuraminate Pyruvate-lyase, and Neu5Ac-permease (hypothetical) in Clostridium perfringens. [Key , sialoglycoprotein , free sialic acid f—at>, ...

On the basis of this anti-proteolytic effect of sialic acids, a hypothetical model435 for the role of sialidase in clostridial infections is shown in Scheme 4. It is considered that the bacterial enzyme releases sialic acids from cell-surface glycoproteins of the infected tissue, which thereafter can be readily attacked by proteases. This cooperation between sialidase and protease may support the spreading of the bacteria. Acylneuraminate pyruvate-lyase, also shown in this model, degrades sialic acids for energy supply, and growth, of the bacteria. 

C. Aug6, S. David, C. Gautlteron, A. Malleron, and B. Cavayd, Preparation of six naturally occurring sialic acids with immobilized acylneuraminate pyruvate lyase, New J. Chem. 72 733 (1988). 

C. Aug6, B. Bouxom, B. Cavayd, and C. Gautheron, Scope and limitations of the aldol condensation catalyzed by immobilized acylneuraminate pyruvate lyase, Tetrahedron Lett. 30 2217 (1989). 

Condensations with pyruvate in the presence of V-acylneuraminate pyruvate lyase immobilized on agarose. 

As shown in Fig. 13, sialic acids liberated by sialidase are degraded by the aldolase systematically named sialate (acylneuraminate)-pyruvate lyase (EC 4.1.3.3), resulting in... 

The most specific method for sialic acid determination is possible with enzymes. Using acylneuraminate pyruvate-lyase (EC 4.1.3.3), sialic acids are cleaved to acylmannosamines and pyruvate. By coupling this reaction to lactate dehydrogenase the reduction of pyruvate allows monitoring of the pyruvate concentration and drives the lyase reaction, otherwise an equilibrium reaction, to completion (Brunetti et al. 1963). The concentration of sialic acid can be measured by spectrophotometry or fluorimetry of the amount of NADH oxidized on completion of the reaction, or by measurement of the initial rate of NADH oxidation. A less sensitive alternative is determination of N-acylmannosamine using colorimetric methods. Brunetti et al. (1962) utilized the Morgan-Elson reaction with dimethyl-aminobenzaldehyde for this purpose. 

A modification of the acylneuraminate pyruvate-lyase assay using pyruvate oxidase to generate H2O2, and measurement of this with peroxidase and p-chlorophenol-4-aminoantipyrine has been described (Sugahara et al. 1980). The measurement of the chromophore is at 505 nm and a molar extinction coefficient of 1.14 X 104 was found at this wavelength. This test has been used in conjunction with sialidase for estimation of serum sialic acid (Sugahara et al. 1980), but remains less sensitive than the method coupled with lactate dehydrogenase. 

Fig. 6. Proposed mechanism of reversible cleavage of Neu5Ac by acylneuraminate pyruvate-lyase (from Nees et al 1976).

Acylneuraminate pyruvate-lyase has been demonstrated in hog kidney (Brunetti et al. 1962), beef kidney (Sirbasku and Binkley 1970), mouse (NOhle and Schauer 1981), rat intestine (Witt-Krause et al. 1979) and human liver (unpublished). Studies on the uptake of NeuSAc and NeuSGc in the mouse and rat have provided evidence that an acylneuraminate pyruvate-lyase is active in most tissues utilizing free sialic acid (NOhle and Schauer 1981). 

The cellular localization of the acylneuraminate pyruvate-lyase has not been identified. Human liver, fractionated by classical ultracentrifugation methods, showed activity only in the 100,000 g supernatant (unpublished). 

Which occurs naturally in a trout glycoprotein, has been made by condensation of D-mannose with pyruvic acid in the presence of immobilized acylneuraminate pyruvate lyase. D-Lyxose and 2-deoxy-... 

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