The formation of a reversible adsorption complex between a dimer of N-acetyl-L-tyrosine [di-(N-acetyl-ʟ-tyrosine), (NAT)2] and horseradish peroxidase (HRP) compound II (CII) was demonstrated using a kinetic approach. A specific K value (0.58 mM) was deduced for this step from stopped-flow measurements. The dimerization of the dipeptide Gly-Tyr was analysed at the steady state and compared with (NAT)2 dimerization [(NAT)2 → (NAT)4]. A saturation of the enzyme was observed for both substrates within their range of solubility. In each case the rate of dimerization reflected the rate-limiting step of compound II reduction to the native HRP (E) (k/KkII → E). The k values for (Gly-Tyr)2 and (NAT)4 formation were 254 s-1 and 3.6 s-1 respectively. The K value of Gly-Tyr was 24 mM. It was observed that the value (0.7 mM) for (NAT)2 was close both to its specific K value for the second step of reduction (CII → E) and to its thermodynamic dissociation constant (Kd = 0.7 mM) with the resting form of the enzyme. As (NAT)2 was a tighter ligand but a poorer substrate than Gly-Tyr, a steady-state kinetic study was performed in the presence of both substrates. A kinetic model which includes an enzyme-substrate adsorption prior to each of the two steps of reduction was derived. This one agreed reasonably well with the experimental data.

This content is only available as a PDF.