Analysis of essential histidine residues of maize branching enzymes by chemical modification and site-directed mutagenesis |
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| Authors: | Kazumi Funane Nathalie Libessart Douglas Stewart Toru Michishita and Jack Preiss |
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| Institution: | (1) Department of Biochemistry, Michigan State University, 48824 East Lansing, Michigan;(2) Present address: National Food Research Institute, 2-1-2 Kannondai, Tsukuba, 305 Ibaraki, Japan;(3) Present address: Department of Plant Science, Waite Agricultural Research Institute, University of Adelaide, 5064 Glen Osmond, SA, Australia;(4) Present address: Agricultural Research Institute, Hokuren Federation of Agricultural Cooperatives E7, N6, 060 Higashi-ku, Sapporo, Japan |
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| Abstract: | Incubation of maize branching enzyme, mBEI and mBEII, with 100 μM diethylpyrocarbonate (DEPC) rapidly inactivated the enzymes.
Treatment of the DEPC-inactivated enzymes with 100–500 mM hydroxylamine restored the enzyme activities. Spectroscopic data
indicated that the inactivation of BE with DEPC was the result of histidine modification. The addition of the substrate amylose
or amylopectin retarded the enzyme inactivation 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
were individually replaced by Ala via site-directed mutagenesis to probe their role in catalysis. Expression of these mutants
inE. coli showed a significant decrease of the activity and the mutant enzymes hadK
m values 10 times higher than the wild type. Therefore, residues His320 and His508 do play an important role in substrate binding. |
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| Keywords: | Branching enzyme chemical modification diethyl pyrocarbonate site-directed mutagenesis histidine residues substrate binding |
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