Site-directed mutagenesis evidence for arginine-384 residue at the active site of maize branching enzyme II.
Essential arginine residues are suggested to be located at the active sites of maize branching enzymes (BE) based on the evidence that two arginine residues are conserved in all BE from various species and that as little as one arginine residue is located at the active site of maize BE by phenylglyoxal (PGO) modification. To determine the exact location of the active-site arginine residue in BE, we employed peptide mapping and site-directed mutagenesis approaches. A single trypsin-digested, [14C]PGO-labeled peptide was purified from maize BEII by two rounds of HPLC separation, but we failed to obtain amino acid sequencing information. Site-directed mutagenesis was then used to create one mutant (arginine-384 to alanine-384), R384A. Immunoblotting result showed that BEII protein was expressed at a similar level in the wild type and the R384A mutant. However, BE activity in the R384A mutant was only 1.4% of the wild type. These results support the conclusion that the conserved arginine-384 residue is important in BEII catalysis.
Department of Biochemistry, Michigan State University, East Lansing 48824, USA.
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D015061:1,4-alpha-Glucan Branching Enzyme / Q000737:chemistry*; D000595:Amino Acid Sequence; D000688:Amylose / Q000378:metabolism; D001120:Arginine / Q000737:chemistry*; D001665:Binding Sites; D017124:Conserved Sequence; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D015870:Gene Expression; D006863:Hydrogen-Ion Concentration; D015151:Immunoblotting; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010449:Peptide Mapping / Q000379:methods; D010658:Phenylglyoxal / Q000494:pharmacology; D010940:Plant Proteins / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry; D014170:Transformation, Genetic; D003313:Zea mays / Q000737:chemistry
D010940:Plant Proteins; D011994:Recombinant Proteins; D000688:Amylose; D001120:Arginine; D015061:1,4-alpha-Glucan Branching Enzyme; C116400:starch-branching enzyme IIb; D010658:Phenylglyoxal