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 microM diethylpyrocarbonate (DEPC) rapidly inactivated the enzymes. Treatment of the DEPC-inactivated enzymes with 100500 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 in E. coli showed a significant decrease of the activity and the mutant enzymes had Km values 10 times higher than the wild type. Therefore, residues His320 and His508 do play an important role in substrate binding.
Funane
K
K
Department of Biochemistry, Michigan State University, East Lansing 48824, USA.
Libessart
N
N
Stewart
D
D
Michishita
T
T
Preiss
J
J
D015061:1,4-alpha-Glucan Branching Enzyme / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics; D000595:Amino Acid Sequence; D017124:Conserved Sequence; D017931:DNA Primers / Q000378:metabolism; D004047:Diethyl Pyrocarbonate / Q000494:pharmacology; D004926:Escherichia coli; D006639:Histidine / Q000032:analysis*; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009841:Oligonucleotides / Q000378:metabolism; D013056:Spectrophotometry, Ultraviolet; D013329:Structure-Activity Relationship; D003313:Zea mays / Q000201:enzymology*
D017931:DNA Primers; D009841:Oligonucleotides; D006639:Histidine; D015061:1,4-alpha-Glucan Branching Enzyme; D004047:Diethyl Pyrocarbonate