I.G. Gazaryan et al. / Phytochemistry 51 (1999) 175±186
181
Comparisons of the catalytic activities of iso-
enzymes are usually based on their speci®c activities.
However, this approach assumes a linear dependence
of the catalytic rate on the enzyme concentration. In
the case of the peroxidase catalysed degradation of
IAA, the comparison is complicated by the occurrence
of both enzymatic reactions (Eqs. (1), (2) and (6)±(8))
and non-enzymatic radical reactions (Eqs. (3)±(5)).
The situation is illustrated in Fig. 4 with a pronounced
eect of PNP-C concentration on the composition and
rates of production of IAA degradation products. The
rate of IAA degradation depends linearly on the
enzyme concentration only at comparatively high con-
centrations of PNP-C (>0.4 mM) when no lag-period
can be detected under the experimental conditions
detectable intermediate does not necessarily mean a
lower activity for a given peroxidase catalysing the
oxygenase reaction. The detection of compound III,
although indicative of a particular reaction mechanism
(
as de®ned by its sensitivity to catalase), does not
necessarily mean a higher rate of IAA oxidative degra-
dation. The low catalytic activity of PNP-C in the oxy-
genase reaction and the clear detection of compound
III in the rapid scan transient kinetic experiments can
be rationalised by consideration of the recently solved
crystal structures for PNP-C (Schuller, Ban, van
Huystee, McPherson, & Poulos, 1996) and HRP-C
(
Gajhede, Schuller, Henriksen, Smith, & Poulos, 1997).
Although the structural elements of PNP-C and HRP-
C are essentially identical the loop insertion between
helixes F and G that is characteristic of class III per-
oxidases is shorter in PNP-C (27 residues) than in
HRP-C (34 residues). This loop region is also highly
variable amongst class III peroxidases and de®nes part
of the substrate access channel to the haem edge and
is thought to be at least in part responsible for sub-
strate speci®city. In the case of HRP-C, this insertion
contains Phe179 which is involved in binding aromatic
substrates (Smith, & Veitch, 1998). In addition, PNP-
C has Gly at this position with the Phe shifted by one
residue. PNP-C has a glycosylation site at Asn60
located at the exposed end of the loop while the near-
est potential glycosylation site in HRP-C is Asn 57
positioned to one side of the loop. These structural
dierences may decrease the accessibility to the haem-
binding pocket of PNP-C for skatole hydroperoxide
and therefore favour compound III over compound I
and II formation at the initiation step. Compound III
although more easily formed, is less active than com-
pound II, resulting in a lower catalytic activity for
PNP-C in the oxidative degradation of IAA.
(
rates of 0.2, 2 and 6.5 mM/min per 0.07, 0.40 and 1.4
mM PNP-C, respectively, Fig. 4A). At low enzyme
concentrations (<0.4 mM) the initial rate of skatole
hydroperoxide production (0.2 mM/min, Fig. 4B) is
equal to that of IAA degradation (Fig. 4A) but the
®
nal enzymatic conversion to indole-3-methanol is
only 5% compared to 20% at the higher enzyme con-
centrations (Fig. 4D). The products of the non-enzy-
matic degradation of skatole hydroperoxide, e.g.
oxindole-3-carbinol and 3-methylene-oxindole, make a
major contribution to the IAA degradation products
at low enzyme concentrations. At high enzyme concen-
trations (0.4 and 1.4 mM), the rates of skatole hydro-
peroxide degradation (1.6 and 5.0 mM/min,
respectively) and indole-3-methanol production (0.4
and 1.3 mM/min, respectively) are proportional to the
enzyme concentration. We conclude that skatole
hydroperoxide is the primary reaction product and it
is subsequently enzymatically reduced to indole-3-
methanol.
Comparison of the rates of indole-3-aldehyde pro-
duction (Fig. 4C) with those for skatole hydroperoxide
(
Fig. 4B) and indole-3-methanol production (Fig. 4D)
demonstrate that the overall contribution of the
indole-3-aldehyde production route increased with
increasing enzyme concentration. Therefore, indole-3-
aldehyde is unlikely to be a degradation product of
skatole hydroperoxide. Indole-3-aldehyde production
is thought to be catalysed by ferrous enzyme, however,
the mechanism is still unclear (Gazarian et al., 1998).
Since the rate of formation and relative amounts of
IAA degradation products depend on enzyme concen-
tration, we have chosen to compare the catalytic activi-
ties of dierent peroxidases at those enzyme
concentrations that yield equal rates of IAA degra-
dation. Both isozymes of peanut peroxidase were less
active than either TOP or HRP-C. The initial rates of
IAA degradation were equal to 7.5 mM/min as cata-
lysed by 1.4 mM PNP-C, 0.7 mM PNP-A, 0.05 mM
TOP and 0.07 mM HRP-C under the above experimen-
tal conditions. Thus, the absence of compound III as a
In order to understand in more detail the mechan-
ism of compound III formation, we studied the eect
of SOD in the presence and absence of catalase on the
kinetics and product distribution during IAA degra-
dation (Fig. 5). Any rate enhancement observed in the
presence of SOD resulting from hydrogen peroxide
production formed by superoxide radical dismutation,
should be decreased by the co-addition of catalase and
SOD. An enhanced rate of IAA degradation was still
observed in the presence of SOD and catalase Fig. 5.
This observation is consistent with our proposal that a
ternary complex involving peroxidase, IAA and oxy-
gen is initially formed that subsequently releases super-
oxide anion and IAA cation radicals (Eqs. (1) and (2)).
In control experiments 1 h incubation with SOD
resulted in the appearance of traces (<0.1%) of oxi-
ndole-3-carbinol and indole-3-aldehyde (not shown).
Gazaryan, Irina G.
