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Title: On the intermediacy of carboxyphosphate in biotin-dependent carboxylations

Abstract

In the ATP-dependent carboxylation of biotin that is catalyzed by most biotin-dependent carboxylases, a fundamental mechanistic question is whether the ATP activates bicarbonate (via the formation of carboxyphosphate as an intermediate) or whether the ATP activates biotin (via the formation of O-phosphobiotin). The authors have resorted to three mechanistic tests using the biotin carboxylase subunit of acetyl-CoA carboxylase from Escherichia coli: positional isotope exchange, intermediate trapping, and {sup 18}O tracer experiments on the ATPase activity. First, no catalysis of positional isotope exchange in adenosine 5{prime}-(({alpha},{beta}-{sup 18}O,{beta},{beta}-{sup 18}O{sub 2})triphosphate) was observed when either biotin or bicarbonate was absent, nor was any exchange seen in the presence of both N-1-methylbiotin and bicarbonate. Second, the putative carboxyphosphate intermediate could not be trapped as its trimethyl ester, under conditions of incubation and analysis where the authentic triester was shown to be adequately stable. In the third test, however, they showed that the ATPase activity of biotin carboxylase that is seen in the absence of biotin, an activity that is known to parallel the normal carboxylase reaction when biotin is present, occurs with the transfer of an {sup 18}O label directly from ({sup 18}O)bicarbonate into the product P{sub i}. This result suggests that the bicarbonate-dependentmore » biotin-independent ATPase reaction catalyzed by biotin carboxylase goes via carboxyphosphate and that the carboxylation of biotin itself may proceed analogously.« less

Authors:
 [1];  [2]
  1. Sankyo Co., Ltd., Tokyo (Japan)
  2. Harvard Univ., Cambridge, MA (USA)
Publication Date:
OSTI Identifier:
6941059
Resource Type:
Journal Article
Journal Name:
Biochemistry; (USA)
Additional Journal Information:
Journal Volume: 27:21; Journal ID: ISSN 0006-2960
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; ATP-ASE; ISOTOPIC EXCHANGE; BIOTIN; CARBOXYLATION; CARBOXYLASE; BIOLOGICAL PATHWAYS; ACID CARBONATES; ATP; BIOCHEMICAL REACTION KINETICS; ESCHERICHIA COLI; OXYGEN 18; ACID ANHYDRASES; AZOLES; BACTERIA; CARBON-CARBON LYASES; CARBOXY-LYASES; CARBOXYLIC ACIDS; CHEMICAL REACTIONS; ENZYMES; EVEN-EVEN NUCLEI; HETEROCYCLIC ACIDS; HETEROCYCLIC COMPOUNDS; HYDROLASES; IMIDAZOLES; ISOTOPES; KINETICS; LIGHT NUCLEI; LYASES; MICROORGANISMS; NUCLEI; NUCLEOTIDES; ORGANIC ACIDS; ORGANIC COMPOUNDS; ORGANIC NITROGEN COMPOUNDS; ORGANIC SULFUR COMPOUNDS; OXYGEN ISOTOPES; PHOSPHOHYDROLASES; REACTION KINETICS; STABLE ISOTOPES; VITAMIN B GROUP; VITAMINS; 550201* - Biochemistry- Tracer Techniques

Citation Formats

Ogita, Takeshi, and Knowles, J R. On the intermediacy of carboxyphosphate in biotin-dependent carboxylations. United States: N. p., 1988. Web. doi:10.1021/bi00421a009.
Ogita, Takeshi, & Knowles, J R. On the intermediacy of carboxyphosphate in biotin-dependent carboxylations. United States. doi:10.1021/bi00421a009.
Ogita, Takeshi, and Knowles, J R. Tue . "On the intermediacy of carboxyphosphate in biotin-dependent carboxylations". United States. doi:10.1021/bi00421a009.
@article{osti_6941059,
title = {On the intermediacy of carboxyphosphate in biotin-dependent carboxylations},
author = {Ogita, Takeshi and Knowles, J R},
abstractNote = {In the ATP-dependent carboxylation of biotin that is catalyzed by most biotin-dependent carboxylases, a fundamental mechanistic question is whether the ATP activates bicarbonate (via the formation of carboxyphosphate as an intermediate) or whether the ATP activates biotin (via the formation of O-phosphobiotin). The authors have resorted to three mechanistic tests using the biotin carboxylase subunit of acetyl-CoA carboxylase from Escherichia coli: positional isotope exchange, intermediate trapping, and {sup 18}O tracer experiments on the ATPase activity. First, no catalysis of positional isotope exchange in adenosine 5{prime}-(({alpha},{beta}-{sup 18}O,{beta},{beta}-{sup 18}O{sub 2})triphosphate) was observed when either biotin or bicarbonate was absent, nor was any exchange seen in the presence of both N-1-methylbiotin and bicarbonate. Second, the putative carboxyphosphate intermediate could not be trapped as its trimethyl ester, under conditions of incubation and analysis where the authentic triester was shown to be adequately stable. In the third test, however, they showed that the ATPase activity of biotin carboxylase that is seen in the absence of biotin, an activity that is known to parallel the normal carboxylase reaction when biotin is present, occurs with the transfer of an {sup 18}O label directly from ({sup 18}O)bicarbonate into the product P{sub i}. This result suggests that the bicarbonate-dependent biotin-independent ATPase reaction catalyzed by biotin carboxylase goes via carboxyphosphate and that the carboxylation of biotin itself may proceed analogously.},
doi = {10.1021/bi00421a009},
journal = {Biochemistry; (USA)},
issn = {0006-2960},
number = ,
volume = 27:21,
place = {United States},
year = {1988},
month = {10}
}