Biochemical Journal

Research article

A normalized plot as a novel and time-saving tool in complex enzyme kinetic analysis

Ignacio G. BRAVO, Félix BUSTO, Dolores de ARRIAGA, Miguel A. FERRERO, Leandro B. RODRÍGUEZ-APARICIO, Honorina MARTÍNEZ-BLANCO, Ángel REGLERO

Abstract

A new data treatment is described for designing kinetic experiments and analysing kinetic results for multi-substrate enzymes. Normalized velocities are plotted against normalized substrate concentrations. Data are grouped into n+1 families across the range of substrate or product tested, n being the number of substrates plus products assayed. It has the following advantages over traditional methods: (1) it reduces to less than a half the amount of data necessary for a proper description of the system; (2) it introduces a self-consistency checking parameter that ensures the ‘scientific reliability’ of the mathematical output; (3) it eliminates the need for a prior knowledge of Vmax; (4) the normalization of data allows the use of robust and fuzzy methods suitable for managing really ‘noisy’ data; (5) it is appropriate for analysing complex systems, as the complete general equation is used, and the actual influence of effectors can be typified; (6) it is amenable to being implemented as a software that incorporates testing and electing among rival kinetic models.

  • data normalization
  • enzymology
  • model–fitting
  • parameter estimation
  • rate equations