An increase in apparent affinity for sucrose of mung bean sucrose synthaseis caused by in vitro phosphorylation or directed mutagenesis of Ser(11)

A mutational analysis of mung bean (Vigna radiata Wilczek) sucrose synthase was performed by site-directed mutagenesis of the recombinant protein expr essed in Escherichia coli, in which two different acidic amino acid residue s (Asp or Glu) were introduced at Ser(11) (S11D, S11E), Only the wild-type enzyme (Ser(11)) was phosphorylated in vitro by a Ca2+-dependent protein ki nase from soybean root nodules, suggesting that this is the specific target residue in mung bean sucrose synthase, The apparent affinity for sucrose w as increased in this phosphorylated enzyme and also in the S11D and S11E mu tant enzymes, although the affinities for UDP-glucose and fructose were sim ilar in the wild-type, phosphorylated wild-type, and mutant enzymes. These results suggest that a monoanionic (1(-)) side chain at position 11 mimics the Ser(11)-P2- residue to bind and cleave sucrose for the synthesis of UDP -glucose. Since the S11E mutant enzyme showed the lowest K-m (sucrose) and the highest catalytic efficiency of the recombinant proteins, the enzymic p roperties of this S11E mutant were further characterized, The results showe d that replacement of Ser(11) with Glu(11) modestly protected the sucrose s ynthesis activity against phenolic glycosides and altered the enzyme nucleo tide specificity. We postulate that the introduction of negative charge at Ser(11) is possibly involved in the enzymatic perturbation of sucrose synth ase.