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-IB-spHI, in which the C-termin al 229 amino acids of maize endosperm branching enzyme isoform II (mBEII) a re replaced by the corresponding 284 amino acids of isoform I (mBEI), sugge sted that the carboxyl terminus of maize branching enzymes may be involved in catalytic efficiency and substrate preference. In the present study, add itional hybrids of mBEI and mBEII were generated and expressed in Escherich ia 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 l evels of V-max in branching activity, K-m for reduced amylose AS 320, and s ubstrate preference for amylose than amylopectin when compared to mBEII-IBs pHI. 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 comple te loss of activity. Replacement of the deleted C-terminal additional 87 am ino 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 mBEI I-IBspHI is required for maximum catalytic efficiency. Another C-terminal r egion, residues Gln510-Asp648, of mBEII-IBspHI (Gln476-Asp614 of mBEI) may be involved in substrate-preference determination. (C) 2000 Academic Press.