EPIMERIZATION VIA CARBON-CARBON BOND-CLEAVAGE - L-RIBULOSE-5-PHOSPHATE 4-EPIMERASE AS A MASKED CLASS-II ALDOLASE

Studies indicating that the E. coli L-ribulose-5-phosphate 4-epimerase employs an ''aldolase-like'' mechanism are reported. This NAD(+)-inde pendent enzyme epimerizes a steseocenter that does not bear an acidic proton and therefore it cannot utilize a simple deprotonation-reproton ation mechanism. Sequence similarities between the epimerase and the c lass II L-fuculose-1-phosphate aldolase suggest that the two may be ev olutionarily related and that the epimerization may occur via carbon-c arbon bond cleavage and re-formation. Conserved residues thought to pr ovide the metal ion ligands of the epimerase have been modified using site-directed mutagenesis. The resulting mutants show low k(cat) value s in addition to a reduced affinity for Zn2+. These observations serve to establish that there is a structural link between between the acti ve site geometry of the epimerase and the aldolase. In addition, the H 97N mutant was found to catalyze the condensation of dihydroxyacetone and glycolaldehyde phosphate to produce a mixture of L-ribulose-5-phos phate and D-xylulose-5-phosphate. This observation of aldolase activit y establishes that the epimerase active site is capable of promoting c arbon-carbon bond cleavage. Furthermore, glycolaldehyde phosphate was shown to be a competitive inhibitor of the mutant enzyme (K-t = 0.37 m M) but not of the wild-type enzyme. The mutation apparently causes the epimerase to become ''leaky'' and enables it to bind/generate the nor mal reaction intermediates from the unbound aldol cleavage products.