MATHEMATICAL-MODEL FOR THE DETERMINATION OF ENZYME-ACTIVITY BASED ON ENZYMATIC AMPLIFICATION BY SUBSTRATE CYCLING

A mathematical description of an enzyme amplification mechanism involv ing the coupling of the enzymatic reaction which is to be measured to a specific substrate cycle is presented, obtaining equations that allo w the system to become linear in the steady-state. The amplification a pproach has been applied to the continuous measurement of pyruvate kin ase activity. The enzymes L-lactate dehydrogenase and L-lactate oxidas e are used to cycle the pyruvate product of pyruvate kinase catalytic activity to L-lactate, with the concomitant disappearance of one molec ule of beta-NADH in each turn of the cycle, which is monitored spectro photometrically at 340 nm. A comparison is made with the standard line ar coupled assay. This shows that any improvement obtained is dependen t on the activity of the coupling enzymes, increasing with time. The m odel is simple and it can be applied to the amplified measurement of a ny enzyme whose catalytic reaction product may be recycled.