Cinètica quìmica

www.biochemj.org
Biochem. J. (2009) 417, 269–275 (Printed in Great Britain) doi:10.1042/BJ20080690

269

Enzymes or redox couples? The kinetics of thioredoxin and glutaredoxin reactions in a systems biology context
Ch´ S. PILLAY, Jan-Hendrik S. HOFMEYR, Brett G. OLIVIER, Jacky L. SNOEP and Johann M. ROHWER1 e
Triple-J Group for Molecular Cell Physiology, Department of Biochemistry,Stellenbosch University, ZA-7602 Matieland, Stellenbosch, South Africa

Systems biology approaches, such as kinetic modelling, could provide valuable insights into how thioredoxins, glutaredoxins and peroxiredoxins (here collectively called redoxins), and the systems that reduce these molecules are regulated. However, it is not clear whether redoxins should be described as redox couples (with redoxpotentials) or as enzymes (with Michaelis– Menten parameters) in such approaches. We show that in complete redoxin systems, redoxin substrate saturation and other purported enzymatic behaviours result from limitations in the redoxin redox cycles in these systems. Michaelis–Menten parameters are

therefore inappropriate descriptors of redoxin activity; data from redoxin kinetic experiments shouldrather be interpreted in terms of the complete system of reactions under study. These findings were confirmed by fitting kinetic models of the thioredoxin and glutaredoxin systems to in vitro datasets. This systems approach clarifies the inconsistencies with the descriptions of redoxins and emphasizes the roles of redoxin systems in redox regulation. Key words: glutathione, kinetic modelling, NADPH,redoxin, reductase, thiol.

INTRODUCTION

Thioredoxins, glutaredoxins and peroxiredoxins (which we collectively refer to as redoxins) are components of systems that are involved in a number of key redox reactions in vivo [1–7]. The thioredoxin system consists of thioredoxin and thioredoxin reductase, and the glutaredoxin system consists of glutaredoxin, glutathione and glutathione reductase(Figure 1). In these systems, reducing equivalents are transferred from NADPH to oxidized substrates via one or more coupled redox cycles. The roles of these systems in redox regulation are now being considered in a systems biology context [8]. A first step in any systems biology approach, such as kinetic modelling, is to define clearly the components under study. In the case of the redoxins, it is notimmediately apparent whether they should be modelled as redox couples or as enzymes. Redox potentials and ratios of oxidized to reduced redoxin have been used to describe redoxins in vivo and in vitro [2,9–11], suggesting that redoxins should be modelled as redox couples. This approach has been adopted for modelling thioredoxins in a number of quantitative descriptions of the cellular redoxenvironment [12–14]. On the other hand, in assays involving complete redoxin systems (Figure 1), redoxins have been quantified with Michaelis– Menten kinetic parameters [15–18], suggesting that they should be modelled as enzymes. Indeed, evidence has been presented that redoxins are Ping Pong enzymes (see for example [18–20]). There are, however, a number of inconsistencies in the description of redoxinsas enzymes. For example, in contrast with the catalytic cycles of conventional enzymes, thioredoxins become inactive (oxidized) after reacting with their substrates (Figure 1). A separate enzyme-catalysed reaction is required to return thioredoxin to its active form. Other inconsistencies include a varying turnover number for the reduction of insulin by thioredoxin (compare with Table 1 in [15]),glutaredoxin substrates that do not have ‘true’ K M values [20], Michaelis–Menten

parameters that underestimate the activity of yeast peroxiredoxins [21] and peroxiredoxins that do not show substrate saturation behaviour (see for example [22]). Thus, any systems biology analysis of these redoxin systems is complicated firstly by the description of the redoxins themselves, and secondly by the...