Mechanistic deductions from multiple kinetic and solvent deuterium isotope effects and pH studies of pyridoxal phosphate dependent carbon-carbon lyases: escherichia coli tryptophan indole-lyase
Analysis of the pH dependence of the kinetic parameters and competitive inhibitor Ki values for tryptophan indole-lyase suggests two enzymic groups must be unprotonated in order to facilitate binding and catalysis of tryptophan. The V/K for tryptophan and the pKi for oxindolyl-L-alanine, a putative transition state analogue and competitive inhibitor, decrease below two pK values of 7.6 and 6.0, while the Ki for L-alanine, also a competitive inhibitor, is 3300-fold larger (20 mM) than that for oxindolyl-L-alanine and increases below a single pK of 7.6. A single pK of 7.6 is also observed in the V/K profile for the alternate substrate, S-methyl-L-cysteine. Therefore, the enzymic group with a pK of 7.6 is responsible for proton abstraction at the 2-position of tryptophan, while the enzymic group with a pK of 6.0 interacts with the indole portion of tryptophan and probably catalyzes formation of the indolenine tautomer of tryptophan (in concert with proton transfer to C-3 of indole from the group with pK 7.6) to facilitate carbon-carbon bond cleavage and elimination of indole. The pH variation of the primary deuterium isotope effects for proton abstraction at the 2-position of tryptophan (DV = 2.5 and D(V/Ktrp) = 2.8) are pH independent, while the Vmax for tryptophan or S-methyl-L-cysteine is the same and also pH independent. Thus, substrates bind only to the correctly protonated form of the enzyme. Further, tryptophan is not sticky, and the pK values observed in both V/K profiles are the correct ones.
- Research Organization:
- Univ. of Wisconsin, Madison (USA)
- OSTI ID:
- 6407683
- Journal Information:
- Biochemistry; (United States), Vol. 27:19
- Country of Publication:
- United States
- Language:
- English
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Related Subjects
DEUTERIUM
ISOTOPE EFFECTS
LYASES
BIOCHEMICAL REACTION KINETICS
CATALYSIS
CYSTEINE
ESCHERICHIA COLI
PH VALUE
TRACER TECHNIQUES
TRYPTOPHAN
AMINO ACIDS
AROMATICS
AZAARENES
AZOLES
BACTERIA
CARBOXYLIC ACIDS
ENZYMES
HETEROCYCLIC ACIDS
HETEROCYCLIC COMPOUNDS
HYDROGEN ISOTOPES
INDOLES
ISOTOPE APPLICATIONS
ISOTOPES
KINETICS
LIGHT NUCLEI
MICROORGANISMS
NUCLEI
ODD-ODD NUCLEI
ORGANIC ACIDS
ORGANIC COMPOUNDS
ORGANIC NITROGEN COMPOUNDS
ORGANIC SULFUR COMPOUNDS
PYRROLES
REACTION KINETICS
STABLE ISOTOPES
THIOLS
550201* - Biochemistry- Tracer Techniques