On the role of histidine and tyrosine residues in E. coli asparaginase. Chemical modification and 1H-nuclear magnetic resonance studies.
The relative importance of tyrosine and histidine residues for the catalytic action of Escherichia coli asparaginase (L-asparagine amidohydrolase, EC 3.5.1.1) was studied by chemical modification and 1H-NMR spectroscopy. We show that, under appropriate reaction conditions, N-bromosuccinimide (NBS) as well as diazonium-1H-tetrazole (DHT) inactivate by selectively modifying two tyrosine residues per asparaginase subunit without affecting histidyl moieties. We further show that diethyl pyrocarbonate (DEP), a reagent considered specific for histidine, also modifies tyrosine residues in asparaginase. Thus, inactivation of the enzyme by DEP is not indicative of histidine residues being involved in catalysis. In 1H-nuclear magnetic resonance (NMR) spectra of asparaginase signals from all three histidine residues were identified. By measuring the pH dependencies of these resonances, pKa values of 7.0 and 5.8 were derived for two of the histidines. Titration with aspartate which tightly binds to the enzyme at low pH strongly reduced the signal amplitude of the pKa 7 histidyl moiety as well as those of resonances of one or more tyrosine residues. This suggests that tyrosine and histidine are indeed constituents of the active site.
Bagert
U
U
Institute of Physiological Chemistry, Philipps University Marburg Lahn, F.R.G.
Röhm
K H
KH
D001215:Asparaginase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D001224:Aspartic Acid / Q000378:metabolism; D001974:Bromosuccinimide / Q000494:pharmacology; D055598:Chemical Phenomena; D002621:Chemistry; D004047:Diethyl Pyrocarbonate / Q000494:pharmacology; D004926:Escherichia coli / Q000201:enzymology*; D006639:Histidine; D009682:Magnetic Resonance Spectroscopy; D013053:Spectrophotometry; D013777:Tetrazoles / Q000494:pharmacology; D014443:Tyrosine
D013777:Tetrazoles; C007314:5-diazo-1H-tetrazole; D001224:Aspartic Acid; D014443:Tyrosine; D006639:Histidine; D001215:Asparaginase; D001974:Bromosuccinimide; D004047:Diethyl Pyrocarbonate