Syoichi Makimoto

Pressure effects on substrate activation phenomena in the α-chymotrypsin-catalyzed hydrolysis of p-nitrophenyl acetate

With and without p-chlorophenol as an activator, the rates of hydrolysis of p-nitrophenyl acetate catalyzed by alpha-chymotrypsin were measured at pressures up to 2 kbar at 25 degrees C. From the pressure dependence of the rate constant (kcat)A and (kcat)0 of the product formation with and without an activator, the activation volumes (delta V not equal to cat)A and (delta not equal to cat)0 were +2 and -6 +/- 1 cm3.mol-1. From the pressure dependence of the equilibrium constant (KA) of incorporation of p-chlorophenol into the enzyme, the volume change (delta VA) was -10 +/- 1 cm3.mol-1. The mechanisms of the substrate activation are discussed in terms of the activation and reaction volumes.

Effect of pressure on the pre-steady-state kinetics of the hydrolysis of anilide substrates catalyzed by α-chymotrypsin

The pre-steady-state of the hydrolysis of succinyl-L-alanyl-L-alanine p-nitroanilide (SA2pNA) and succinyl-L-alanine p-nitroanilide (SApNA) catalyzed by alpha-chymotrypsin in Tris buffer solution was measured up to 1.5 kbar at 25 degrees C. The pressure dependence of the formation of tetrahedral intermediate from the Michaelis complex on the rate constant (k1) gave activation volumes, delta V1 not equal to, of -33 +/- 2 cm3 X mol-1 for SA2pNA and -30 +/- 2 cm3 X mol-1 for SApNA. The reaction mechanism of alpha-chymotrypsin is discussed in terms of the activation volumes.

Pressure Effects on Thermal Inactivation of Taka-amylase A

Pressure effects on the thermal inactivation of Taka-amylase A (TAA) have been studied by the measurement of the rate of hydrolysis of p-nitrophenyl-α-maltoside (pNαM) catalyzed by TAA in the range of 25–75°C up to 2 kbar. At each pressure, the apparent rate of hydrolysis increases with temperature and the rate of hydrolysis decreases against time above a certain temperature. This decrease corresponds to the decrease in enzyme activity that was left by the thermal inactivation of TAA with time. The activation enthalpies, ΔHD≠ and ΔHN≠, for the thermal inactivation and reactivation, and the enthalpy changes, ΔH, for the thermal inactivation are 220 ∼ 170 kJ/mol, −400 ∼ −170 kJ/mol, and 580 ∼ 280 kJ/mol, respectively. The activation volumes, ΔVD≠ and ΔVN≠, for the thermal inactivation and reactivation, and the volume changes, ΔV, for the thermal inactivation are 22 ∼ 30 cm3/mol, −100 ∼ −60 cm3/mol and 120 ∼ 79 cm3/mol, respectively. The mechanisms of the thermal inactivation of TAA are discussed in terms of each thermodynamic parameter.

High Pressure Studies of Enzyme Catalysis

Abstract From the effect of pressure on the Michaelis-Menten kinetics of α-chymotrypsin, the volume changes for the transfer process of substrate molecules to an enzyme and the activation volumes for the catalytic reaction were determined. From the plot of volume profiles against the reaction coordinate for each elementary process, the reaction mechanism of α-chymotrypsin was identified as a single-proton-transfer mechanism, and the high catalytic efficiency/specificity was explained by the volume change for the substrate binding and activation volumes for the hydrolysis.