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A Class I (Schiff Base) Fructose Diphosphate Aldolase of Prokaryotic Origin

PURIFICATION AND PROPERTIES OF MICROCOCCUS AEROGENES ALDOLASE

From the ¹ From the Department of Biochemistry, University of Washington, Seattle, Washington 98105

In contrast to all prokaryotic organisms previously studied, the fructose-P₂ cleavage activity of crude Micrococcus aerogenes extracts was found to be insensitive to high levels of EDTA and o-phenanthroline. This observation suggested that M. aerogenes contained a Class I (Schiff base) fructose-P₂ aldolase instead of the metallo (Class II)-aldolase typically found in other prokaryotes. The enzyme was purified about 1000-fold with 70% recovery. The purified aldolase, which appears essentially homogeneous by electrophoretic and gel filtration criteria, was completely active in the presence of 0.01 m EDTA and o-phenanthroline. In addition, direct Zn⁺⁺ analysis on a "metal-free" preparation showed that a fully active enzyme sample contained only 0.01 g atom of Zn⁺⁺ per mole. In contrast to Class II aldolases, the M. aerogenes enzyme lost activity on incubation with the substrate dihydroxyacetone-P in the presence of NaBH₄. The degree of enzyme inactivation was directly correlated with the amount of [³H]dihydroxyacetone-P covalently bound to enzyme after treatment with NaBH₄ and a stoichiometry of one dihydroxyacetone-P binding site per enzyme subunit was calculated. Identification of the substrate binding site as a lysyl residue by amino acid analysis establishes that this fructose-P₂ aldolase is a Class I enzyme.

The general catalytic features of the M. aerogenes enzyme resemble those of other Class I aldolases. (a) Its activity is not enhanced by the presence of K⁺. (b) It cleaves fructose-1-P at a significant rate (fructose-P₂ to fructose-1-P activity ratio = 3.3). (c) It has a broad pH optima for fructose-P₂ cleavage and a narrow optima for fructose-1-P cleavage; both pH profiles are similar to those of rabbit aldolases A and C. (d) The kinetic parameters K_m and V_max for the two substrates are very similar to those of rabbit aldolases. K_m (fructose-P₂) = 1 x 10^-6 m; K_m (fructose-1-P) = 1 x 10^-2 m; V_max (fructose-P₂) = 8.2 units per mg; V_max (fructose-1-P) = 2.4 units per mg. The enzyme, however, differs from other Class I aldolases in that its fructose-P₂ cleavage activity is completely insensitive to digestion with carboxypeptidase A and its activity is unaffected by treatment with sulfhydryl reagents.

M. aerogenes aldolase is distinct from all aldolases previously studied in that it is composed of a single polypeptide chain.

The detection of only Class II aldolase activity in other micrococcus strains (Micrococcus lactilyticus, Micrococcus lysodeikticus, and Acidaminococcus fermentans) as well as all previous studies on the fructose-P₂ aldolases of prokaryotic cells, suggest that the Class I aldolase described here has a very restricted distribution among the bacteria.

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