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Title: Carbon-13 and deuterium isotope effects on the catalytic reactions of biotin carboxylase

Abstract

/sup 13/C and /sup 2/H kinetic isotope effects have been used to investigate the mechanism of enzymic biotin carboxylation. /sup D/(V/K) is 0.50 in 80% D/sub 2/O at pD 8.0 for the forward reaction and 0.57 at pD 8.5 for the phosphorylation of ADP by carbamoyl phosphate. These values approach the theoretical maximum limit for a reaction in which a proton is transferred from a sulfhydryl to a nitrogen or oxygen base. Therefore, it appears that this portion of the reaction is at or near equilibrium. /sup 13/(V/K) at pH 8 is 1.007; the small magnitude of this number suggests that the reaction is almost fully committed by the time the carbon-sensitive steps are reached. There does not appear to be a reverse commitment to the reaction under the conditions in which /sup 13/(V/K) was determined. A large forward commitment is consistent with the failure to observe positional isotope exchange from the ..beta gamma..-bridge position to the ..beta..-nonbridge position in (/sup 18/O/sub 4/)ATP or washout of /sup 18/O from the ..gamma..-nonbridge positions. Transfer of /sup 18/O from bicarbonate to inorganic phosphate in the forward reaction was clearly observed, however. These observations suggest that biotin carboxylase exists in two distinct formsmore » which differ in the protonation states of the two active-site bases, one of which is a sulfhydryl. Only when the sulfhydryl is ionized and the second base protonated can catalysis take place. Carboxylation of biotin is postulated to occur via a pathway in which carboxyphosphate is formed by nucleophilic attack of bicarbonate on ATP. Decarboxylation of carboxyphosphate in the active site generates CO/sub 2/, which serves to carboxylate the isourea tautomer of biotin that is generated by the removal of the proton on N1' by the ionized sulfhydryl.« less

Authors:
;
Publication Date:
Research Org.:
Univ. of Wisconsin, Madison (USA)
OSTI Identifier:
6735094
Resource Type:
Journal Article
Journal Name:
Biochemistry; (United States)
Additional Journal Information:
Journal Volume: 27:12
Country of Publication:
United States
Language:
English
Subject:
62 RADIOLOGY AND NUCLEAR MEDICINE; CARBOXYLASE; BIOCHEMICAL REACTION KINETICS; ISOTOPE EFFECTS; NUCLEAR MAGNETIC RESONANCE; ACID CARBONATES; ATP; BIOTIN; CARBON 13; CARBOXYLATION; HEAVY WATER; OXYGEN 18; PHOSPHORUS 31; SODIUM COMPOUNDS; ALKALI METAL COMPOUNDS; AZOLES; CARBON ISOTOPES; CARBON-CARBON LYASES; CARBOXY-LYASES; CARBOXYLIC ACIDS; CHEMICAL REACTIONS; ENZYMES; EVEN-EVEN NUCLEI; EVEN-ODD NUCLEI; HETEROCYCLIC ACIDS; HETEROCYCLIC COMPOUNDS; HYDROGEN COMPOUNDS; IMIDAZOLES; ISOTOPES; KINETICS; LIGHT NUCLEI; LYASES; MAGNETIC RESONANCE; NUCLEI; NUCLEOTIDES; ODD-EVEN NUCLEI; ORGANIC ACIDS; ORGANIC COMPOUNDS; ORGANIC NITROGEN COMPOUNDS; ORGANIC SULFUR COMPOUNDS; OXYGEN COMPOUNDS; OXYGEN ISOTOPES; PHOSPHORUS ISOTOPES; REACTION KINETICS; RESONANCE; STABLE ISOTOPES; VITAMIN B GROUP; VITAMINS; WATER; 550601* - Medicine- Unsealed Radionuclides in Diagnostics

Citation Formats

Tipton, P A, and Cleland, W W. Carbon-13 and deuterium isotope effects on the catalytic reactions of biotin carboxylase. United States: N. p., 1988. Web. doi:10.1021/bi00412a020.
Tipton, P A, & Cleland, W W. Carbon-13 and deuterium isotope effects on the catalytic reactions of biotin carboxylase. United States. doi:10.1021/bi00412a020.
Tipton, P A, and Cleland, W W. Tue . "Carbon-13 and deuterium isotope effects on the catalytic reactions of biotin carboxylase". United States. doi:10.1021/bi00412a020.
@article{osti_6735094,
title = {Carbon-13 and deuterium isotope effects on the catalytic reactions of biotin carboxylase},
author = {Tipton, P A and Cleland, W W},
abstractNote = {/sup 13/C and /sup 2/H kinetic isotope effects have been used to investigate the mechanism of enzymic biotin carboxylation. /sup D/(V/K) is 0.50 in 80% D/sub 2/O at pD 8.0 for the forward reaction and 0.57 at pD 8.5 for the phosphorylation of ADP by carbamoyl phosphate. These values approach the theoretical maximum limit for a reaction in which a proton is transferred from a sulfhydryl to a nitrogen or oxygen base. Therefore, it appears that this portion of the reaction is at or near equilibrium. /sup 13/(V/K) at pH 8 is 1.007; the small magnitude of this number suggests that the reaction is almost fully committed by the time the carbon-sensitive steps are reached. There does not appear to be a reverse commitment to the reaction under the conditions in which /sup 13/(V/K) was determined. A large forward commitment is consistent with the failure to observe positional isotope exchange from the ..beta gamma..-bridge position to the ..beta..-nonbridge position in (/sup 18/O/sub 4/)ATP or washout of /sup 18/O from the ..gamma..-nonbridge positions. Transfer of /sup 18/O from bicarbonate to inorganic phosphate in the forward reaction was clearly observed, however. These observations suggest that biotin carboxylase exists in two distinct forms which differ in the protonation states of the two active-site bases, one of which is a sulfhydryl. Only when the sulfhydryl is ionized and the second base protonated can catalysis take place. Carboxylation of biotin is postulated to occur via a pathway in which carboxyphosphate is formed by nucleophilic attack of bicarbonate on ATP. Decarboxylation of carboxyphosphate in the active site generates CO/sub 2/, which serves to carboxylate the isourea tautomer of biotin that is generated by the removal of the proton on N1' by the ionized sulfhydryl.},
doi = {10.1021/bi00412a020},
journal = {Biochemistry; (United States)},
number = ,
volume = 27:12,
place = {United States},
year = {1988},
month = {6}
}