アスパラギン酸アミノ基転移酸素の精密反応機構の研究

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タイトル別名
  • Refined Reaction Mechanism of Aspartate Aminotransferase
  • アスパラギン酸アミノ基転移酵素の精密反応機構の研究
  • アスパラギンサン アミノキ テンイ コウソ ノ セイミツ ハンノウ キコウ ノ ケンキュウ

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Although the reaction catalyzed by aspartate aminotransferase (AAT) has been extensively studied and the Karpeisky-Ivanov mechanism has been widely accepted, there remain many unsolved important problems. These are: 1) Acid-base chemistry of AAT has not been established because of the unusual kinetic behavior at low pH. 2) The exceptionally low pK_a of the Schiff base of AAT makes its reaction mechanism unapplicable to other B_6 enzymes. 3) The role of the enzyme protein in catalysis has not yet been clarified. This review summarizes our recent work that gave solutions to these problems. The rate of disappearance of the Schiff base-protonated enzyme (E_LH^+) upon reaction with aspartate was apparently slower than that of the Schiff base-unprotonated enzyme (E_L). At low pH and low substrate concentrations, this difference was found to come from the relatively slow exchange of proton between the PLP-Lys258 Schiff base and the solvent. With increasing pH and aspartate concentration, the rate of disappearance of E_LH^+ became larger than taht expected from the model in which EL is the sole reactive species. The deviation was excellently explained by assuming the association of E_LH^+ and aspartate with unprotonated amino group (S) in addition to the association of E_L and SH^+. The newly discovered route (E_LH^+-S) seems to be the main route in B_6 enzymes which have only the E_LH^+ structure. The establishment of the pH study in the transient kinetics of AAT enabled us to analyze the reaction of AAT with a substrate analogue 2-methylaspartate on a wide range of pH. The result showed that the Schiff bases in the Michaelis complex and the external aldimine complex were partially protonated, irrespective of the solvent pH. The intrinsic pK_a values of the Schiff base, estimated from the fraction of the protonation, was found to increase progressively from the unliganded enzyme to the external aldimine complex. A novel model for the modulation of the Schiff base pK_a, in which the imine-pyridine torsion of the Schiff base regulates the basicity of the Schiff base, was proposed and was supported by the spectroscopic study on the Lys258-side-chain-fragmented AAT. The strain of the protonated Schiff base is interpreted to enhance the catalytic ability of the enzyme by increasing the energy level of the free enzyme plus substrate at neutral pH relative to the transition state. Combining these observations altogether, a refined reaction mechanism of AAT was proposed.

収録刊行物

  • ビタミン

    ビタミン 73 (3), 135-147, 1999

    公益社団法人 日本ビタミン学会

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