Abstract
Abstract The CO2 hydration activity of human carbonic anhydrase B is rapidly inactivated by 1-fluoro-2,4-dinitrobenzene at neutral pH. The inactivation reaction is second order and is retarded by the presence of various enzyme inhibitors. The second order rate constant for inactivation is about 100 times the second order rate constant for the reaction of 1-fluoro-2,4-dinitrobenzene and α-N-acetylhistidine. The inactivated enzyme was purified and was found to contain one 2,4-dinitrophenyl group per molecule, at the 3' position of a histidine residue. While such a reacted enzyme has lost more than 95% of its CO2 hydration activity, it retains over half its esterase activity. The dinitrophenyl group can be quantitatively removed by thiolysis, regenerating a fully active native carbonic anhydrase. Dinitrophenyl carbonic anhydrase binds iodoacetate with somewhat greater affinity than the unmodified enzyme. However, iodoacetate does not react covalently with the dinitrophenyl enzyme, as it does with the native enzyme. Analysis of the tryptic peptides of dinitrophenyl carbonic anhydrase shows that reaction takes place with histidine residue 204, the same residue modified by iodoacetate. It is suggested that histidine 204, while clearly in the active site, may not play any direct role in enzyme catalysis. Modifications at this position may disrupt the normal substrate binding site.
Highlights
MethodsChemicals-All chemicals were reagent grade and obtained commercially. Bio-Rex-70, 200400 mesh, and Aminex Q15S are products of Bio-Rad Laboratories. 14C-FDNB was purchased from Nuclear Supplies, Inc., Encino, California (specific activity1.61 mCi per mmole) or from Amersham/SearleCorporation, Des Plaines, Illinois (specific activity 31.5 mCi per mmole).Bis-(2-hydroxymethyl)imino-tris(hydroxymethyl)methane was obtained from General Biochemicals, Inc
The dinitrophenyl group can be quantitatively removed by thiolysis, regenerating a fully active native carbonic anhydrase
In this paper we present the results of a study of the chemical modification of human carbonic anhydrase B by FDNB, which reacts rapidly with the imidazole ring of 1 histidine residue per enzyme molecule
Summary
Chemicals-All chemicals were reagent grade and obtained commercially. Bio-Rex-70, 200400 mesh, and Aminex Q15S are products of Bio-Rad Laboratories. 14C-FDNB was purchased from Nuclear Supplies, Inc., Encino, California (specific activity1.61 mCi per mmole) or from Amersham/SearleCorporation, Des Plaines, Illinois (specific activity 31.5 mCi per mmole).Bis-(2-hydroxymethyl)imino-tris(hydroxymethyl)methane was obtained from General Biochemicals, Inc. Chemicals-All chemicals were reagent grade and obtained commercially. Bio-Rex-70, 200400 mesh, and Aminex Q15S are products of Bio-Rad Laboratories. 14C-FDNB was purchased from Nuclear Supplies, Inc., Encino, California 1.61 mCi per mmole) or from Amersham/Searle. Corporation, Des Plaines, Illinois (specific activity 31.5 mCi per mmole). Bis-(2-hydroxymethyl)imino-tris(hydroxymethyl)methane was obtained from General Biochemicals, Inc. Acetazolamide was a gift from the American Cyanamid Company. Trypsin (treated with diisopropyl phosphofluoridate) with a specific activity of 160 units per mg was purchased from Worthington Biochemical Corporation. Lauryl trimethylammonium bromide was obtained from K and K Laboratories
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