Probing the location and function of the conserved histidine residue of phosphoglucose isomerase by using an active site directed inhibitor N-bromoacetylethanolamine phosphate.
Phosphoglucose isomerase (EC 5.3.1.9) catalyzes the interconversion of D-glucopyranose-6-phosphate and D-fructofuranose-6-phosphate by promoting an intrahydrogen transfer between C1 and C2. A conserved histidine exists throughout all phosphoglucose isomerases and was hypothesized to be the base catalyzing the isomerization reaction. In the present study, this conserved histidine, His311, of the enzyme from Bacillus stearothermophilus was subjected to mutational analysis, and the mutational effect on the inactivation kinetics by N-bromoacetylethanolamine phosphate was investigated. The substitution of His311 with alanine, asparagine, or glutamine resulted in the decrease of activity, in k(cat)/K(M), by a factor of 10(3), indicating the importance of this residue. N-bromoacetylethanolamine phosphate inactivated irreversibly the activity of wild-type phosphoglucose isomerase; however, His311 --> Ala became resistant to this inhibitor, indicating that His311 is located in the active site and is responsible for the inactivation of the enzyme by this active site-directed inhibitor. The pKa of His311 was estimated to be 6.31 according to the pH dependence of the inactivation. The proximity of this value with the pKa value of 6.35, determined from the pH dependence of k(cat)/K(M), supports a role of His311 as a general base in the catalysis.
Meng
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Graduate Institute of Agricultural Biotechnology, National Chung Hsing University, Taichung, Taiwan, Republic of China. mhmeng@dragon.nchu.edu.tw
Chane
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Sun
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Hsiao
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D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D017360:Arabidopsis / Q000201:enzymology; D001665:Binding Sites; D017124:Conserved Sequence; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000494:pharmacology*; D004926:Escherichia coli / Q000201:enzymology; D004983:Ethanolamines / Q000737:chemistry / Q000494:pharmacology*; D001411:Geobacillus stearothermophilus / Q000201:enzymology*; D005956:Glucose-6-Phosphate Isomerase / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism*; D006639:Histidine; D006801:Humans; D007700:Kinetics; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D017355:Zymomonas / Q000201:enzymology
D004791:Enzyme Inhibitors; D004983:Ethanolamines; D011994:Recombinant Proteins; D006639:Histidine; C020161:N-bromoacetylethanolamine phosphate; D005956:Glucose-6-Phosphate Isomerase