Ae. Johnson et Me. Tanner, EPIMERIZATION VIA CARBON-CARBON BOND-CLEAVAGE - L-RIBULOSE-5-PHOSPHATE 4-EPIMERASE AS A MASKED CLASS-II ALDOLASE, Biochemistry, 37(16), 1998, pp. 5746-5754
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.