Solvent and solvent proton dependent steps in the galactose oxidase reaction
Solvent and solvent proton dependent steps involved in the mechanism of the enzyme galactose oxidase have been examined. The deuterium kinetic solvent isotope effect (KSIE) on the velocity of the galactose oxidase catalyzed oxidation of methyl beta-galactopyranoside by O/sub 2/ was measured. Examination of the thermodynamic activation parameters for the reaction indicated that the isotope effect was attributable to a slightly less favorable delta H value, consistent with a KSIE on proton transfer. A detailed kinetic analysis was performed, examining the effect of D/sub 2/O on the rate of reaction over the pH range 4.8-8.0. Both pL-rate profiles exhibited bell-shaped curves. Substitution of D/sub 2/O as solvent shifted the pKes values for the enzymic central complex: pKes1 from 6.30 to 6.80 and pKes2 from 7.16 to 7.35. Analysis of the observed shifts in dissociation constants was performed with regard to potential hydrogenic sites. pKes1 can be attributed to a histidine imidazole, while pKes2 is tentatively assigned to a Cu2+-bound water molecule. A proton inventory was performed (KSIE = +1.55); the plot of kcat vs. mole fraction D/sub 2/O was linear, indicating the existence of a single solvent-derived proton involved in a galactose oxidase rate-determining step (or steps). The pH dependence of CN- inhibition was also examined. The Ki-pH profile indicated that a group ionization, with pKa = 7.17, modulated CN- inhibition; Ki was at a minimum when this group was in the protonated state. The inhibition profile followed the alkaline limit of the pH-rate profile for the enzymic reaction, suggesting that the group displaced by CN- was also deprotonating above pH 7.
- Research Organization:
- State Univ. of New York, Buffalo
- OSTI ID:
- 5727486
- Journal Information:
- Biochemistry; (United States), Journal Name: Biochemistry; (United States) Vol. 26:12; ISSN BICHA
- Country of Publication:
- United States
- Language:
- English
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Related Subjects
59 BASIC BIOLOGICAL SCIENCES
BARYONS
BIOCHEMICAL REACTION KINETICS
BIOLOGICAL PATHWAYS
CYANIDES
DEUTERIUM
ELEMENTARY PARTICLES
ENERGY TRANSFER
ENZYMES
FERMIONS
HADRONS
HYDROGEN COMPOUNDS
HYDROGEN ISOTOPES
ISOTOPE APPLICATIONS
ISOTOPE EFFECTS
ISOTOPES
KINETICS
LIGHT NUCLEI
NUCLEI
NUCLEONS
ODD-ODD NUCLEI
OXIDOREDUCTASES
OXYGEN COMPOUNDS
PROTONS
REACTION KINETICS
SOLVENTS
STABLE ISOTOPES
TRACER TECHNIQUES
WATER