title: Alkaline phosphatase is an almost perfect enzyme.
abstract: The second-order rate constant, kcat/km, for catalysis of the hydrolysis of 4-nitrophenyl phosphate by alkaline phosphatase decreases with increasing viscosity in the presence of sucrose or arabinose, with a slope of delta[kcat/Km)0/(kcat/Km)]/delta(eta/eta 0) = 1.4 at pH 8.0, 25 degrees C. This is consistent with rate-limiting diffusional encounter of the substrate with active enzyme and indicates that alkaline phosphatase is a "perfect enzyme". However, the reported second-order rate constants of kcat/Km = 6.6 x 10(6) to 4.6 x 10(7) M-1 s-1 are smaller than the diffusional limit; this shows that only approximately 0.1-1% of the diffusional encounters are productive. The first-order rate constant, kcat, for rate-limiting hydrolysis of the phosphoenzyme intermediate at pH = 6 with saturating substrate concentration is independent of viscosity in aqueous sucrose solutions. This shows that sucrose does not destabilize the transition state for phosphoenzyme hydrolysis. However, at pH 8.0 product dissociation is rate limiting and kcat decreases with increasing viscosity in the presence of sucrose, with slopes of delta(k0/kobsd)/delta(eta/eta 0) = 1.2 in 0.04 M Mops buffer, 1.0 in 0.1 M Tris, and 1.2 in 0.67 M Tris buffer. This is consistent with rate-limiting diffusional separation of inorganic phosphate and of Tris phosphate from the enzyme. In contrast, glycerol causes a large decrease in kcat/Km at pH 8.0 and also decreases kcat at pH 6. This shows that glycerol decreases the rate by a solvent effect on the catalytic activity of the enzyme, as well as by increasing the viscosity.
authors.affiliation: Graduate Department of Biochemistry, Brandeis University, Waltham, Massachusetts 02254-9110.
authors.forename: T T
authors.initials: TT
authors.lastname: Simopoulos
authors.forename: W P
authors.initials: WP
authors.lastname: Jencks
mesh_terms: D000469:Alkaline Phosphatase / Q000737:chemistry / Q000378:metabolism*; D001089:Arabinose; D004058:Diffusion; D004795:Enzyme Stability; D005990:Glycerol; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D066298:In Vitro Techniques; D007700:Kinetics; D009596:Nitrophenols; D009943:Organophosphorus Compounds; D012996:Solutions; D012997:Solvents; D013395:Sucrose; D014783:Viscosity
chemical_list: D009596:Nitrophenols; D009943:Organophosphorus Compounds; D012996:Solutions; D012997:Solvents; C008644:nitrophenylphosphate; D013395:Sucrose; D001089:Arabinose; D000469:Alkaline Phosphatase; D005990:Glycerol