Abstract
The Lineweaver-Burk plot or Hanes-Woolf plot is usually used to determine enzyme kinetics parameters, such as Km, in one-substrate reaction. However, that is often applied to twosubstrate reactions as a conventional or simplified method, because of the complexity of the nonlinear least-squares method and the difficulty in obtaining the appropriate computer program. The reliability of the enzyme kinetics parameters in two-substrate reactions determined by the simplified method was evaluated using the optimized reaction rates equation calculated by a computer program “ASNOP”. The reaction rates of aspartate aminotransferase (AST) and γ-glutamyltransferase (γ-GT) were determined throughout a wide range of substrate concentrations. The rate of AST estimated by the simplified method corresponded well with that calculated by the optimized method under the analytical condition recommended by JSCC. However, a discrepancy in the rate was observed between the two methods at high concentrations of 2-oxoglutarate, because substrate inhibition is neglected in the simplified method. The reaction rate of γ-GT as the complex Ping- Pong bibi mechanism is unreliable when estimated by the simplified method. Furthermore, when the contour lines were drawn by the response surface method (RSM), a large error was observed at regions deviating from a defined midpoint.
