T. Kuriki et al., CONSTRUCTION OF CHIMERIC ENZYMES OUT OF MAIZE ENDOSPERM BRANCHING ENZYME-I AND ENZYME-II - ACTIVITY AND PROPERTIES, The Journal of biological chemistry, 272(46), 1997, pp. 28999-29004
Branching enzyme I and II isoforms from maize endosperm (mBE I and mBE
II, respectively) have quite different properties, and to elucidate t
he domain(s) that determines the differences, chimeric genes consistin
g of part mBE I and part mBE II were constructed. When expressed under
the control of the T7 promoter in Escherichia coli, several of the ch
imeric enzymes were inactive, The only fully active chimeric enzyme wa
s mBE II-I BspHI, in which the carboxyl-terminal part of mBE II was ex
changed for that of mBE I at a BspHI restriction site and was purified
to homogeneity and characterized, Another chimeric enzyme, mBE I-II H
indIII, in which the amino-terminal end of mBE II was replaced with th
at of mBE I, had very little activity and was only partially character
ized, The purified mBE II-I BspHI exhibited higher activity than wild-
type mBE I and mBE II when assayed by the phosphorylase a stimulation
assay. mBE II-I BspHI had substrate specificity (preference for amylos
e rather than amylopectin) and catalytic capacity similar to mBE I, de
spite the fact that only the carboxyl terminus was from mBE I, suggest
ing that the carboxyl terminus may be involved in determining substrat
e specificity and catalytic capacity, In chain transfer experiments, m
BE II-I BspHI transferred more short chains (with a degree of polymeri
zation of around 6) in a fashion similar to mBE II, In contrast, mBE I
-II HindIII transferred more long chains (with a degree of polymerizat
ion of around 11-12), similar to mBE I, suggesting that the amino term
inus of mBEs may play a role in the size of oligosaccharide chain tran
sferred, This study challenges the notion that the catalytic centers f
or branching enzymes are exclusively located in the central portion of
the enzyme; it suggests instead that the amino and carboxyl termini m
ay also be involved in determining substrate preference, catalytic cap
acity, and chain length transfer.