ANALYSIS OF ESSENTIAL HISTIDINE-RESIDUES OF MAIZE BRANCHING ENZYMES BY CHEMICAL MODIFICATION AND SITE-DIRECTED MUTAGENESIS

Incubation of maize branching enzyme, mBEI and mBEII, with 100 mu M di ethylpyrocarbonate (DEPC) rapidly inactivated the enzymes. Treatment o f the DEPC-inactivated enzymes with 100-500 mM hydroxylamine restored the enzyme activities. Spectroscopic data indicated that the inactivat ion of BE with DEPC was the result of histidine modification. The addi tion of the substrate amylose or amylopectin retarded the enyzme inact ivation by DEPC, suggesting that the histidine residues are important for substrate binding. In maize BEII, conserved histidine residues are in catalytic regions 1 (His320) and 4 (His508). His320 and His508 wer e individually replaced by Ala via site-directed mutagenesis to probe their role in catalysis. Expression of these mutants in E. coli showed a significant decrease of the activity and the mutant enzymes had K-m values 10 times higher than the wild type. Therefore, residues His320 and His508 do play an important role in substrate binding.