Investigation into the nature of substrate binding to the dipyrromethane cofactor of Escherichia coli porphobilinogen deaminase
The formation of the dipyrromethane cofactor of Escherichia coli porphobilinogen deaminase was shown to depend on the presence of 5-aminolevulinic acid. A hemA/sup -/ mutant formed inactive deaminase when grown in the absence of 5-aminolevulinic acid since this strain was unable to biosynthesize the dipyrromethane cofactor. The mutant formed normal levels of deaminase, however, when grown in the presence of 5-aminolevulinic acid. Porphobilinogen, the substrate, interacts with the free ..cap alpha..-position of the dipyrromethane cofactor to give stable enzyme-intermediate complexes. Experiments with regiospecifically labeled intermediate complexes have shown that, in the absence of further substrate molecules, the complexes are interconvertible by the exchange of the terminal pyrrole ring of each complex. The formation of enzyme-intermediate complexes is accompanied by the exposure of a cysteine residue, suggesting that substantial conformational changes occur on binding substrate. Specific labeling of the dipyrromethane cofactor by growth of the E. coli in the presence of 5-amino(5-/sup 14/C)levulinic acid has confirmed that the cofactor is not subject to catalytic turnover. Experiments with the ..cap alpha..-substituted substrate analogue ..cap alpha..-bromoporphobilinogen have provided further evidence that the cofactor is responsible for the covalent binding of the substrate at the catalytic site. On the basis of these cummulative findings, it has been possible to construct a mechanistic scheme for the deaminase reaction involving a single catalytic site which is able to catalyze the addition or removal of either NH/sub 3/ or H/sub 2/O. The role of the cofactor both as a primer and as a means for regulating the number of substrates bound in each catalytic cycle is discussed.
- Research Organization:
- Univ. of Southampton (England)
- OSTI ID:
- 6208751
- Journal Information:
- Biochemistry; (United States), Vol. 27:25
- Country of Publication:
- United States
- Language:
- English
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CARBON 14 COMPOUNDS
UPTAKE
ESCHERICHIA COLI
ENZYME ACTIVITY
LYASES
CONFORMATIONAL CHANGES
AMINOLEVULINIC ACID
MUTANTS
SUBSTRATES
WATER
AMINO ACIDS
BACTERIA
CARBOXYLIC ACIDS
ENZYMES
HYDROGEN COMPOUNDS
LABELLED COMPOUNDS
MICROORGANISMS
ORGANIC ACIDS
ORGANIC COMPOUNDS
OXYGEN COMPOUNDS
550201* - Biochemistry- Tracer Techniques