but with a different reductant, the mycobacterial thioredoxin C. Clearly, the exchange of one physiological for another homologous thioredoxin results in a kinetic pattern that seems not to comply with an enzyme substitution mechanism: The lines corresponding to the lower co-substrate concentrations are no longer parallel, suggesting a central complex mechanism, while the ones for higher co-substrate concentrations are parallel, which complies with the expected enzyme substitution mechanism. Further, the lines are no longer straight; they steeply decline when approaching the y axis. However, a detailed investigation of MtTPx by means of stopped-flow analysis of partial reactions (see below) and mass spectroscopic identification of catalytic intermediates provided independent compelling evidence that the catalytic mechanism in essence is an enzyme substitution mechanism and essentially the same for both types of thioredoxins (Trujillo et al., 2006).

The molecular basis of the irregularity still remains a matter of speculation. Budde et al. (Budde et al., 2003) described the phenomenon, in terms of the Changeux-Monod model, as indicating a negative cooperativity between the subunits of the oligomeric enzyme (in this case of TXNPx from Trypanosoma brucei), since it changed its quaternary structure in a redox-dependent manner, as has been reported for many Prxs (see Chapter 3). However, for an enzyme in charge of antiox-idant defense such as a TXNPx a negative cooperativity, which implies lower efficiency under conditions of increased demand, does not make much sense, and one should not ignore the possibility that the irregular kinetic patterns simply reflect a certain weakness inherent in the character of catalysis. Not by chance was the phenomenon first reported for the mitochondrial form of TXNPx from Leishmania infantum (LimTXNPx) (Castro et al., 2002). This enzyme, depending on the nature and concentration of the hydroperoxide used, proved to be oxidatively inactivated already during the few minutes required for conventional activity determination, and it is obvious that inactive or less active enzyme forms might have accumulated also during the steady state kinetic measurements and thus have falsified the concentration of fully active enzyme. Oxidative inactivation as such is a problem common to all peroxiredoxins and, with some mammalian Prxs, is already observed at very low concentrations of hydroperoxides (<1 ^M H2O2) (Yang et al., 2002). In most cases the observed inhibitory effect of the oxidizing substrate on Prxs was reported to be irreversible. The inactivation is now primarily ascribed to over-oxidation of the peroxidatic cysteine to a sulfinic acid. Excess hydroperoxide competes with CR for the reaction of CP, when pre-oxidized to the sulfenic acid form, and the rate of inactivation will therefore depend on the rate constant k+3 (Fig. 1) in relation to that of the competing reaction of Eox1 with an ROOH, as well as on the concentrations of the reactants. This interpretation would also comply with the most surprising discrepancy between the data sets shown in Fig. 3. MtTrxB, being a more efficient reductant for MtTPx than MtTrxC (KM for TrxB<< than KM for TrxC; Table 1), would be in a better position to keep the peroxidase preferentially in the reduced state and thereby to prevent the [Eox1]-dependent inactivation and concomitant kinetic irregularities. In case of MtTPx, however, additional modes of oxidative alterations have to be considered. MtTPx is an atypical 2-Cys Prx that does not require its CR to sustain the entire catalytic cycle but nevertheless can form a disulfide bridge between CP and CR which can be reduced by thioredoxins. Also, a disulfide bridge between CP and a non-conserved catalytically irrelevant cysteine could be detected in an enzyme preparation that was oxidized by slightly over-stoichiometric ROOH concentrations in the absence of reductant (Trujillo et al., 2006). Certainly, these different forms of oxidized enzyme will not react identically with each of the physiological reductants. If or to what extent all these different enzyme forms also induce conformational changes with associated alterations of catalytic efficiencies in the context of the oligomeric assemblies as proposed by Budde et al. (2003), is not easily answered by accessible experimental tools. In short, there are plenty of possibilities to explain deviations from an expected kinetic pattern and, taking together kinetic and chemical evidences, an enzyme substitution mechanism identical or similar to that depicted in Fig. 1 is still the most appropriate concept to describe peroxiredoxin catalysis.

To some extent overoxidation of CP appears to occur even during catalysis in vivo. A kinetic analysis of mammalian Prx I inactivation in the presence of a low steady-state level H2O2(< 1 ^M) indicated that Prx I was over-oxidized at a rate of 0.072 % per turnover at 30 °C (Yang et al., 2002). Susceptibility to over-oxidation in 2-Cys Prxs is related to a structural motif, i e. the C-terminal GCLG tail, present in eukaryotic but not in prokaryotic enzymes.3 The motif is supposed to slow down the reaction between the sulfenic acid derivative of CP with CR, thus giving time for the over-oxidation of the sulfenic acid by a second molecule of oxidizing substrate (Wood et al., 2003). This inactivation process cannot be generally considered a weakness of Prx catalysis. It might well be that nature has taken advantage of this possibility to switch off the enzymes in special situations. The appealing hypothesis arose that the C-terminal tail in eukaryotic Prxs is an adaptation allowing them to function as floodgates, keeping resting levels of hydrogen peroxide low, while permitting higher levels for signal transduction (Wood et al., 2003). The description of sulfiredoxins (Biteau et al., 2003; Chang et al., 2004) and sestrins (Budanov et al., 2004), enzymes responsible for sulfinic acid reduction in typical two-cysteine Prxs (Woo et al., 2005) to recover the enzymatic activity, lends further support to the physiological relevance of the inactivation process. The presence of enzymes that specifically facilitate the redox cycling between the thiol and the sulfinic acid forms of peroxiredoxins might represent an important switch by which the activity of these proteins can be adapted to oxidative challenge.

In addition to the coupled assay, steady state kinetic determinations in perox-iredoxins have also been performed by taking advantage of the decrease in the intensity of thioredoxin fluorescence that occurs upon oxidation (Holmgren, 1972). Figure 4, inset, shows the changes in the emission fluorescence spectra that take

3 Thus, procaryotic Prxs, as well as other Prxs devoid of C-terminal tail, are less sensitive to oxidative inactivation. Accordingly, prokaryotes do not contain sulfiredoxin (Biteau, et al., 2003; see also Chapters 3, 4 and 9).

Was this article helpful?

0 0

Post a comment