Cunxin Wang

Thermokinetic models of enzyme-catalyzed reactions in batch and plug-flow reactors

Abstract This paper reports the thermokinetic models of single-substrate, enzyme-catalyzed reactions occurring in batch and plug-flow reactors, respectively. By analyzing the calorimetric curves of these reactions, these models can be used to produce not only the thermodynamic data ( Δ r H m ) but also the kinetic data ( K m and k 2 ). Using a LKB-2107 batch microcalorimeter and an LKB-2277 Bioactivity Monitor, the catalase-catalyzed decomposition of hydrogen peroxide was studied and its molar reaction enthalpy ( Δ r H m ) measured as −88.88±0.6 kJ mol −1 . The Michaelis constant ( K m ) for H 2 O 2 was determined by the batch and plug-flow thermokinetic models to be 5.03±0.18×10 −3 mol dm −3 and (5.27±0.11)×10 −3 mol dm −3 , respectively. The reliability of these models for determination of the thermokinetics of single-substrate, enzyme-catalyzed reactions occurring in these two types of reactors was verified by the experimental results.

Microcalorimetric study of the toxic effect of sodium selenite on the mitochondria metabolism of Carassius auratus liver

The fundamental thermogenesis curves of the metabolic process of liver mitochondria from Carassius auratus and the toxic effect of Na2SeO3 on it were studied by using an LKB-2277 bioactivity monitor, ampoule method, at 28°C. From the thermogenesis curves, the thermokinetic equations were established under different conditions. The kinetics show that a low concentration of Na2SeO3 (1–4 mg/L) had promoting action on the metabolism process of Carassius auratus liver mitochondria, but that a high concentration of Na2SeO3 (8–16 mg/L) inhibited the mitochondria metabolism.

Thermokinetic studies of the product inhibition of single-substrate, enzyme-catalyzed reactions

Abstract A thermokinetic reduced extent method for the product inhibition of single-substrate, enzyme-catalyzed reactions is proposed in this paper. By analyzing the calorimetric curves of these reactions, this method can be conveniently used to calculate both kinetic parameters ( K m , K i and V m ) and molar reaction enthalpy ( Δ r H m ), and to establish the type of product inhibition simultaneously without adding product. The arginase-catalyzed hydrolysis of L-arginine has been studied by microcalorimetry and the product, L-ornithine, has been established as a competitive reversible inhibitor. The kinetic parameters calculated with this method are in agreement with those given in the literature.

Thermokinetic studies of the irreversible inhibition of single-substrate, enzyme-catalyzed reactions

Abstract A thermokinetic ratio method for the irreversible inhibition of single-substrate enzymecatalyzed reactions is proposed in this paper. By analyzing a measured curve this method can be used to calculate the apparent rate constant of the inhibited reaction without letting the reaction go to completion. Using the LKB-2107 batch microcalorimeter, the arginase-catalyzed hydrolysis of L-arginine in the presence of p -chloromercuribenzoate (PCMB) has been studied and PCMB established as an irreversible mixed inhibitor. The second-order rate constants for inhibition of arginase by PCMB in the absence and presence of L-arginine have been determined by this ratio method to be k EI = 94.4 M − s −1 and k ESI = 35.2 M −1 s −1 , respectively, at 298.15 K. Chemical modification with PCMB indicates that arginase contains three reactive cysteinyl residues at most but these residues are not present at the active site of arginase.

Microcalorimetric studies on the product inhibition of hydrolysis of L-arginine catalyzed by bovine liver arginase

A new thermokinetic reduced extent method for the product inhibition of single substrate enzyme-catalyzed reactions is proposed and compared with the traditional initial rate method in this paper. The arginase-catalyzed hydrolysis of L-arginine to L-ornithine and urea was studied at 37°C in 40 mM sodium barbiturate-HCl buffer solution (pH=9.4). Michaelis constant (Km) for arginine and maximum velocity (Vm) of the reaction were determined by initial method and thermokinetic method. The activation of exogenous manganese to this reaction was also studied. The product inhibition constant (KP), which cannot be obtained directly from the initial rate method, was determined by thermokinetic without adding L-ornithine to the reaction system. When the concentration of Mn2+ in cell is 0.1 mM, the enzyme gets its full activity. Incubation arginase with appropriate concentration of Mn2+resulted in increased Vmax and a higher sensitivity of the enzyme to product with no change in the Km for arginine. We suggest that the exogenous manganese ions in solution have just recovered the activity of arginase, which was lost in dissolving and dilution, but no effect on the mechanism of the reaction.

Effects of protein and phosphate buffer concentrations on thermal denaturation of lysozyme analyzed by isoconversional method

ABSTRACT Thermal denaturation of lysozymes was studied as a function of protein concentration, phosphate buffer concentration, and scan rate using differential scanning calorimetry (DSC), which was then analyzed by the isoconversional method. The results showed that lysozyme thermal denaturation was only slightly affected by the protein concentration and scan rate. When the protein concentration and scan rate increased, the denaturation temperature (Tm) also increased accordingly. On the contrary, the Tm decreased with the increase of phosphate buffer concentration. The denaturation process of lysozymes was accelatated and the thermal stability was reduced with the increase of phosphate concentration. One part of degeneration process was not reversible where the aggregation occurred. The other part was reversible. The apparent activation energy (Ea) was computed by the isoconversional method. It decreased with the increase of the conversion ratio (α). The observed denaturation process could not be described by a simple reaction mechanism. It was not a process involving 2 standard reversible states, but a multi-step process. The new opportunities for investigating the kinetics process of protein denaturation can be supplied by this novel isoconversional method.