On the intermediacy of carboxyphosphate in biotin-dependent carboxylations

Journal Article · Tue Oct 18 04:00:00 EDT 1988 · Biochemistry; (USA)

DOI:https://doi.org/10.1021/bi00421a009· OSTI ID:6941059

Ogita, Takeshi ^[1]; Knowles, J R ^[2]

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1. Sankyo Co., Ltd., Tokyo (Japan)
2. Harvard Univ., Cambridge, MA (USA)

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.

• OSTI ID: 6941059
• Journal Information: Biochemistry; (USA), Journal Name: Biochemistry; (USA) Vol. 27:21; ISSN 0006-2960; ISSN BICHA
• Country of Publication: United States
• Language: English

Related Subjects

550201* -- Biochemistry-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
ACID ANHYDRASES
ACID CARBONATES
ATP
ATP-ASE
AZOLES
BACTERIA
BIOCHEMICAL REACTION KINETICS
BIOLOGICAL PATHWAYS
BIOTIN
CARBON-CARBON LYASES
CARBOXY-LYASES
CARBOXYLASE
CARBOXYLATION
CARBOXYLIC ACIDS
CHEMICAL REACTIONS
ENZYMES
ESCHERICHIA COLI
EVEN-EVEN NUCLEI
HETEROCYCLIC ACIDS
HETEROCYCLIC COMPOUNDS
HYDROLASES
IMIDAZOLES
ISOTOPES
ISOTOPIC EXCHANGE
KINETICS
LIGHT NUCLEI
LYASES
MICROORGANISMS
NUCLEI
NUCLEOTIDES
ORGANIC ACIDS
ORGANIC COMPOUNDS
ORGANIC NITROGEN COMPOUNDS
ORGANIC SULFUR COMPOUNDS
OXYGEN 18
OXYGEN ISOTOPES
PHOSPHOHYDROLASES
REACTION KINETICS
STABLE ISOTOPES
VITAMIN B GROUP
VITAMINS