Arginine residue 384 at the catalytic center is important for branching enzyme II from maize endosperm.
Branching enzyme (BE) belongs to the amylolytic family which contains four highly conserved regions. These regions are proposed to play an important role in catalysis as they are thought to be necessary for catalysis and/or binding the substrate. Only one arginine residue was found to be conserved in a catalytic center at the same position in all known sequences of BEs from various species as well as in the alpha-amylase enzyme family. In mBEII, a conserved Arg residue 384 is in catalytic region 2. We have used site-directed mutagenesis of the Arg-384 residue in order to study its possible role in BE. Previous chemical modification studies (H. Cao and J. Preiss, 1996, J. Prot. Chem. 15, 291-304) suggest that it may play a role in substrate binding. Replacement of Arg-384 by Ala, Ser, Gln, and Glu in the active site caused almost total inactivation. However, a conservative mutation of the conserved Arg-384 by Lys resulted in some residual activity, approximately 5% of the wild-type enzyme. The kinetics of the purified mutant R384K enzyme were investigated and no large effect on the Km of the substrate amylose for BE was observed. Thus, these results suggest that conserved Arg residue 384 in mBEII plays an important role in the catalytic function of BEs but may not be directly involved in substrate binding.
Libessart
N
N
Department of Biochemistry, Michigan State University, East Lansing, Michigan, 48824, USA.
Preiss
J
J
D015061:1,4-alpha-Glucan Branching Enzyme / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D019943:Amino Acid Substitution / Q000235:genetics; D001120:Arginine / Q000235:genetics / Q000378:metabolism*; D020134:Catalytic Domain / Q000235:genetics; D017124:Conserved Sequence; D004789:Enzyme Activation / Q000235:genetics; D015870:Gene Expression; D016297:Mutagenesis, Site-Directed; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000378:metabolism; D012639:Seeds / Q000201:enzymology; D003313:Zea mays / Q000201:enzymology*
D011994:Recombinant Proteins; D001120:Arginine; D015061:1,4-alpha-Glucan Branching Enzyme