Carbon-13 and deuterium isotope effects on the catalytic reactions of biotin carboxylase
/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.
- Research Organization:
- Univ. of Wisconsin, Madison (USA)
- OSTI ID:
- 6735094
- Journal Information:
- Biochemistry; (United States), Vol. 27:12
- Country of Publication:
- United States
- Language:
- English
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Related Subjects
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