H. Mizuguchi et al., Reaction mechanism of fructose-2,6-bisphosphatase - A mutation of nucleophilic catalyst, histidine 256, induces an alteration in the reaction pathway, J BIOL CHEM, 274(4), 1999, pp. 2166-2175
A bifunctional enzyme, fructose-6-phosphate,2-kinase/fructose 2,6-bisphosph
atase (Fru-6-P,2-kinase/Fru-2,6-Pase), catalyzes synthesis and degradation
of fructose a,6-bisphosphate (Fru-2,6-P-2). Previously, the rat liver Fru-2
,6-Pase reaction (Fru-2,6-P-2 --> Fru-6-P + P-i) has been shown to proceed
via a phosphoenzyme intermediate with His(258) phosphorylated, and mutation
of the histidine to alanine resulted in complete loss of activity (Tauler,
A., Lin, K., and Pilkis, S. J. (1990) J. Biol. Chem, 265, 15617-15622). In
the present study, it is shown that mutation of the corresponding histidin
e (His(256)) Of the rat testis enzyme decreases activity by less than a fac
tor of 10 with a k(cat) of 17% compared with the wild type enzyme. Mutation
of His(390) (in close proximity to His(256)) to Ala results in a k(cat) of
12.5% compared with the wild type enzyme. Attempts to detect a phosphohist
idine intermediate with the H256A mutant enzyme were unsuccessful, but the
phosphoenzyme is detected in the wild type, H390A, R255A, R305S, and E325A
mutant enzymes. Data demonstrate that the mutation of His(256) induces a ch
ange in the phosphatase hydrolytic reaction mechanism. Elimination of the n
ucleophilic catalyst, H256A, results in a change in mechanism. In the H256A
mutant enzyme, His(390) likely acts as a general base to activate water fo
r direct hydrolysis of the a-phosphate of Fru-2,6-P-2. Mutation of Arg(255)
and Arg(305) suggests that the arginines probably have a role in neutraliz
ing excess charge on the a-phosphate and polarizing the phosphoryl for subs
equent transfer to either His(256) or water. The role of Glu(325) is less c
ertain, but it may serve as a general acid, protonating the leaving 2-hydro
xyl of Fru-2,6-P-2.