Localization of C-terminal domains required for the maximal activity or for determination of substrate preference of maize branching enzymes.
Previous analysis of a chimeric enzyme mBEII-IBspHI, in which the C-terminal 229 amino acids of maize endosperm branching enzyme isoform II (mBEII) are replaced by the corresponding 284 amino acids of isoform I (mBEI), suggested that the carboxyl terminus of maize branching enzymes may be involved in catalytic efficiency and substrate preference. In the present study, additional hybrids of mBEI and mBEII were generated and expressed in Escherichia coli BL21 (DE3) to dissect the structure/function relationships of the C-terminal regions of maize branching enzymes. A truncated form of purified mBEII-IBspHI, which lacks the C-terminal 58 amino acids, retained similar levels of V(max) in branching activity, K(m) for reduced amylose AS 320, and substrate preference for amylose than amylopectin when compared to mBEII-IBspHI. This indicates that the C-terminal extension derived from mBEI is not required for either catalysis or substrate preference. However, deletion of an additional 87 amino acids from the carboxyl terminus resulted in complete loss of activity. Replacement of the deleted C-terminal additional 87 amino acids with the corresponding 79 amino acids from mBEII restored 25% of the mBEII-IBspHI branching activity without altering substrate preference. It thus appears that a C-terminal region encompassing Leu649-Asp735 of mBEII-IBspHI is required for maximum catalytic efficiency. Another C-terminal region, residues Gln510-Asp648, of mBEII-IBspHI (Gln476-Asp614 of mBEI) may be involved in substrate-preference determination.
Hong
S
S
Department of Biochemistry, Michigan State University, East Lansing, Michigan 48824, USA.
Preiss
J
J
D015061:1,4-alpha-Glucan Branching Enzyme / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D000596:Amino Acids / Q000737:chemistry; D015153:Blotting, Western; D002384:Catalysis; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D020033:Protein Isoforms; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000378:metabolism; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D003313:Zea mays / Q000201:enzymology*
D000596:Amino Acids; D020033:Protein Isoforms; D011994:Recombinant Proteins; D015061:1,4-alpha-Glucan Branching Enzyme