Kinetic evidence for the formation of a Michaelis-Menten-like complex between horseradish peroxidase compound II and di-(N-acetyl-L-tyrosine).
The formation of a reversible adsorption complex between a dimer of N-acetyl-L-tyrosine [di-(N-acetyl-L-tyrosine), (NAT)2] and horseradish peroxidase (HRP) compound II (CII) was demonstrated using a kinetic approach. A specific KIIm 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) (kappcat/Kappm approximately kII-->E). The kappcat values for (Gly-Tyr)2 and (NAT)4 formation were 254 s-1 and 3.6 s-1 respectively. The KappM 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 KIIm 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.
Wang
W
W
Department of Cellular and Molecular Pharmacology, University of California San Francisco, Box 0450, San Francisco, CA 94143-0448, USA.
Noël
S
S
Desmadril
M
M
Guéguen
J
J
Michon
T
T
D000327:Adsorption; D002384:Catalysis; D019281:Dimerization; D004305:Dose-Response Relationship, Drug; D004583:Electrons; D005998:Glycine / Q000378:metabolism; D006735:Horseradish Peroxidase / Q000378:metabolism*; D007700:Kinetics; D008024:Ligands; D008956:Models, Chemical; D010084:Oxidation-Reduction; D011108:Polymers / Q000378:metabolism; D012995:Solubility; D013816:Thermodynamics; D017186:Titrimetry; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000378:metabolism
D008024:Ligands; D011108:Polymers; D014443:Tyrosine; C007543:dityrosine; C025787:N-acetyltyrosine; D006735:Horseradish Peroxidase; D005998:Glycine