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

Journal Article · Tue Sep 20 04:00:00 EDT 1988 · Biochemistry; (United States)

DOI:https://doi.org/10.1021/bi00419a024· OSTI ID:6407683

Kiick, D M; Phillips, R S

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), Journal Name: Biochemistry; (United States) Vol. 27:19; ISSN BICHA
• Country of Publication: United States
• Language: English

Related Subjects

550201* -- Biochemistry-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
AMINO ACIDS
AROMATICS
AZAARENES
AZOLES
BACTERIA
BIOCHEMICAL REACTION KINETICS
CARBOXYLIC ACIDS
CATALYSIS
CYSTEINE
DEUTERIUM
ENZYMES
ESCHERICHIA COLI
HETEROCYCLIC ACIDS
HETEROCYCLIC COMPOUNDS
HYDROGEN ISOTOPES
INDOLES
ISOTOPE APPLICATIONS
ISOTOPE EFFECTS
ISOTOPES
KINETICS
LIGHT NUCLEI
LYASES
MICROORGANISMS
NUCLEI
ODD-ODD NUCLEI
ORGANIC ACIDS
ORGANIC COMPOUNDS
ORGANIC NITROGEN COMPOUNDS
ORGANIC SULFUR COMPOUNDS
PH VALUE
PYRROLES
REACTION KINETICS
STABLE ISOTOPES
THIOLS
TRACER TECHNIQUES
TRYPTOPHAN