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For: Proceedings of the Symposium on
Flavins and Flavoproteins
Amsterdam, The Netherlands
June 10-15, 1965
(Publisher:
MAY 5 1963
CONF-650609-1
MASTER
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NADH. Peroxidase *
M, I. DOLIN
Biology Division, Oak Ridge National Laboratory
Oak Ridge, Tennessee
*
Research sponsored by the United States Atomic Energy Commission under
contract with the Union Carbide Corporation.
.
PATENT CLEARANCE OBTAINED. RELEASE TO
THE PUBLIC IS APPROVED. PROCEDURES
ARE ON FILE IN THE RECEIVING SECTION.
.
VR
Send proof to:
Dr. M. I. Dolin
Biology Division
Oak Ridge National Laboratory
Post Office Box Y
Oak Ridge, Tennessee 37831
INTRODUCTION
:
The cytochrome-free microorganism, Streptococcus faecalis, 1001, is
able to catalyze the four electron reduction of molecular oxygen to water
with NADH as the electron donor
f t . Extracts of fermentatively
grown cells catalyze the following reactions.
ܐܙܐ
NADH + ** + 02 - NAD + H2O2
(1)
NADH + H+ + ,0, NAD+ + 2 H20
. (2)
2 NADH + 2 +* + . Og + 2 NAD* + 2 H20
Reaction (2) 18 catalyzed by the flavoprotein, NADH peroxidase
(NADH,1H,02 oxdidoreductase, EC 1.11.1.1) f t. The H20, 18 supplied by the
a incude extrets, this olidase a
an oxidase that carries out reaction (1). H ome 13 demonstrable
under conditions which lead to the inactivation t t of NADH peroxidase
(a high NADH to 1,0, ratio). Such conditions are achieved through the
use of dilute solutions of crude sonic extract in the presence of 3 x 1023 M
NADH. Reaction (3) takes place in the presence of higher concentrations
of one or more approximately 2 to 3 mg of protein per ml).
It may be noted that apparently single enzymes which catalyze reaction
(3) have been isolated from Clostridium perfringens to and from
aerobically grown S. faecalis t t. The olostridial enzyme does not
catalyze the anaerobio peroxidation of NADH.
The present paper will deal in the main with the spectral properties
of a complex formed between NADH peroxidase and pyridine nucleotides,
..
The paper 18 essentially a review of work that had been accorp 21shed by
1959.
}
RESULTS
Properties of NADH Peroxidase
Methods for preparation and assay of the enzymet and for carrying out
· the spectrophotometric experiments dealing with complex formation't have
been described.
NADH peroxidase is found in the cytoplasm of S. faecalis. On osmotic
lysis of protoplasts, 98 % of the peroxidase 18 liberated in a form which
does not sediment at 100,000 x g in 1.5 hr . The enzyme has been
purified approximately 2000 fold, starting from sonic extracts f t . A
brief summary of the properties of the purified enzyme follows. .
(a) Prosthetic group. The enzyme contains 0.66 % FAD, equivalent to a
minimum molecular weight of 120,000. Unfortunately, the small amount of
enzyme available did not allow an electrophoretic determination of the molecular
weight. The FAD 18 completely liberated at 0 in 10 % trichloracetic acid,
or at 100 at pH 7.0. There are no detectable hematin corponents and no
metals are present in amounts equivalent to the bound flavin. The native engyme
is nonfluorescent.
(b) Substrate specificity- NADH is the only known physiological reductant
for the peroxidase activity of the enzyme, however both NADH and NADPH
can serve as donors for the very weak oxidase activity of the enzyme (see
Table 1 ). Ferricyanide, menadione and 1,4-naphthoquinone can function
as electron acceptors.
(o) pH optimum- The peroxidase reaction 18 optimal at pH 5.4, in 0.1 M
sodium acetate buffer.
(d) Activators and inhibitors. The peroxidase is stimulated by anions
of the lower fatty acids (acetate to butyrate) and by high concentrations of
phosphate. Neither 0.01 M CN nor 0.05 M azide are inhibitory. The enzyme
18 stable towards -SH inhibitors such as pochloromerouribenzoate, and arsenite.
*As*, out and Pb are inhibitory. Incubation or the enzyme with NADH, causes
a first order decay of enzyme activity (k = 0.066 min, på 5.4). This ...
Inhibition 18 prevented or arrested, but not reversed by 1 x 10-3 M H2O2. ...
(e) Michaelis Constants and Turnover -(pH 5.4. 26°)- In the peroxddase
reaction, the x for a 1.4 x 10-5 M, the K, for 190, = 2 x 20-4 M .
and the Ver = 6 x 102 moles NADH oxidized per mole FAD per min.
...
;
:
:
.
.
"
I.
..
2
..
in
--
-
-
WWW
i Spectrum of Enzyme-Substrate Complex- Kinetic data suggest that the
mechanism of NADH peroxidation involves est or the formation of a rate
limiting temary complex between enzyme, NADH and H2O2, bythemsequenttad
formation of twombinary complexes. Spectrophotometric evidence for the
formation of a binary complex between NADH and the flavoprotein het been
presented * t, and 46-411ustrated-14(Fig. 1 ). When excess
NADH is added to the flavoprotein, the 450 mi band decreases only about
25 %, this change being accompanied by the formation of a broad absorption
. band centered at about 540 mu. The addition of excess hydrosulate to the
NADH reduced enzyme causes little further change except for a small decrease
in the 450 mi band. In the absence of NADH, however, hydrosulfite causes
typical bleaching of the flavoprotein. 6
C These results imply that NADH combines with the flavoprotein to form
a complex which cannot be dissociated by reducing equivalents from hydrosulfite:
On the addition of H2O2 to substrato-reduced enzyme, the spectrum of the
couplex disappears immediately, to be replaced by the spectrum of the
oxddized flavoprotein.
.......... ... ....---
11


Not all pyridine nucleotides that interact with the enzyme cause the
formation of both the 450 and 540 mu bands. As shown in Fig. 1. NADPH .
causes about the same reduction in intensity of the 450 m band as NADH,
without causing the prod•ction of the 540 rou band. NMNH behaves similarly.
Conversely, reduced pyridine aldehyde hypoxanthine dinucleotide induces the
formation of a longwavelength band, but causes little change in the 450 mu
region. These results show that an absorbance decrease at 450 mi 18 not
obligatorily linked with the formation of a 540 mu band, and quadranta,
Titration of Reduced Enzyme with NAD* analogues- The 450 and 540 ms
bands are regenerated on the addition of either NAD * or NADH to the
fydrosulfite reduced enzyme k eto (The spectra produced in this way are
almost identioal to that formed on the addition of NADH to oddizod enzyme).
.
..
This circumstance makes it possible to titrate hydrosulfite reduced enzyme
. With either reduced or oxidized pyridine nucleotides, by following the
absorbance changes at 450 or 540 m, typical saturation curves are obtained.
Consideration of the possible oxidation-reduction state of the complex will
::be deferred until the Discussion section. It will merely bo mentioned here
that titration of hydrosulfite reduced enzyme seems to give meaningful
results since the dissociation constants calculated from these experiments
can be correlated with Michaels constants.
Inspection of the saturation curves (F48. 2) shows that the physiological
substrate of the enzyme is bound more efficiently than the other analogues
shown. Since NAD* and NADH give virtually the same saturation curve, it must
: be concluded that the regeneration of the 450 mu band is not attributable
to traces of NAD* in the NADH preparation. The fact that NADP" and NMN
give the same saturation curve will be returned to later. Ribosyl
nicotinamide and pyridinealdehyde hypoxanthine dinucleotide cause ong ly
a small regeneration of the 450 m band. Dissociation constants calculated
fror. experiments such as that shown in Fig. are independent of the
enzyme concentration over the range 1,7x10-SM to 7x10-5M (as bound FAD)
and do not seem to be influenced by the hydrosulfite level. When NAD
analogues that cause both the 450 and 540 mu bands are used, the same in
dissociation constants are obtained by using the absorbance changes at .
450 m 540 m. Then two changes are linked in a constant ratio during the
titration. At the end of the titration, if the system is allowed to revert
to the oxidized state, the spectrum returns to that of the oxddized enzyme.
..
.

Extinction coefficients of the 450 and 540 mu Band in the Presence of
Hydrosulfite- Table I lists the millimolar extinction coefficients of the
complexes formed when an excess of a pyridine nucleotide is added to the
· hydrosulfite reduced flavoprotein. These data reveal some of the structural
features that the substrate must posses in order for complexing to cccur.
The amino residue of the carboxamide group can be replaced with either
canboyamide
coH or -CH2 , however simultaneous replacement of the Aamtho group with H
and the adenylic acid amino group with -OH leads to almost complete loss
of ability to form the complex. Both NADP and NMN cause the formation of
the 450 mu band, it neither compound causes the formation of the 540 mu".
band. These results show that the adenylic acid moiety 1s necessary for
formation of the long wavelength band, but not for formation of the 450 me.
suggests that
· band. Practaments the 2' phosphate of the ribosyl adenine moiety of
The fact than
NADP "prevents the binding of the adenylic acid group. TSC NADP+and NMN
give very similar results.) Removal of the 50 phsophate of NMN leads to
virtually complete loss of ability tú generate the 450 mu band. Neither
reduced NAD-acetone (the analogue in which one of the C-4 hydrogen atomg
of NADH is replaced with acetone) nor the primary acid modification
product of NADH (presumably trihydromonohydroxy NAD tot ) are able to
generate the characteristic absorption spectrum of the complexed enzyme.
Mixtures of the individual moieties that go to make up the dinucleotide
structure of NAD do not replace NAD itself as a complexing agent. For
example'
t y, the following mixtures added to hydrosulfite-reduced flavoprotein
cause little effect other than that produced by r18syl nicotinamide or NMN
themselves: nicotinamide + adenosine diphosphate ribose, ribosyl adenine +
ADP, NMN + AMP. The results demonstrate that the complexing ability of
NAD sobiect depends upon an Intact dinucleotide structure. .
.
.
:
: .
Formation of complexes Between Oxddized Enzyme and Reduced Pyridine
Nucleotide- The absorption bands produced on the addition of reduced
pyridine nucleotide to oxidized enzyme have approximately the same intensity
as the bands formed by adding either reduced or oxidized pyridine nucleotide
to hydrosulfite-reduced enzyme. For two of the pyridine nucleotides, however,
the spectra are altered in the presence of hydrosulfite. Comparison of the
extinction coefficients obtained under the conditions of Table I with
those found on the addition of reduced pyridine nucleotide to oxidized
flavoprotein suggest that the complex between reduced pyridinealdehyde
adenine dinucleotide and enzyme is 30 % dissociated by hydrosulfite, whereas
the complex formed between enzyme and pyridinealdehyde hypoxanthine
dinucleotide is 75% dis sociated by hydrosulfite. These results are
reduced
obtained at both the 450 and 540 m bands. The inability of pyridinealdehyde
hypoxanthine dinucleotide to cause much change in the 450 we band of
oxidized flavoprotein (Fig. 1 ) may be another aspect of its inability to
form strong complexes with the enzyme. These results are further correlated
with the fact that pyridinealdehye adenine dimucleotide is a weak substrate
for the enzyme, and that pyridinealdehyde hypoxanthine dinucleotide is
almost totally inactive (next section). The results imply that the
anino groups of the nicotinamide and adenine moieties represent two of
the structural features necessary for the formation of complexes which are
not readily dissociated by chemical reducing agents.
Spectra of the substrate-reduced enzyme were determined with all
the pyridine dinucleotides listed in Table I . The spectra are similar to
that of enzyme reduced by NADH, except for variation in intensity of the
absorption bands and for the fact that the long wave Length band produced
by pyridinealdehyde adenine dinucleotide has its maximum shifted towards 510 mu.
There is no detectable interaction betweon enzyme and either reduced NAD-acetone
or the primary acid modifaoti on product of NADH. Neither of twou HADH
ع
فسمممهمعدهتععطععمعمعمعمعمعمعمعمعمعممهههم
جمعتننبمممممت
These analogues of NADH do not change the spectrum of oxidized flavoprotein
nor do they block the reduction of the flavoprotein by hydrosulfite. .
..'
.
.
.
-
.
.
Comparison of Analogues as Substrates and as Complex Formers- In order to
determine whether the spectral intermediates just described belong to a kinetically
active enzyme species, it would be necessary to evaluate the transient kinetics
for formation and disappearance of the characteristic absorption bands. The
small arount of purified enzyme available precluded sucă studies. As the next
best approach, an attempt was made to see whether any correlation could be
found made between the structures of totoo pyridine nucleotide analogues and their
bath
ability to form complexes with the enzyme and to serve as substrates for
catalytic amounts of enzyme. The results presented in Table I show that
such correlations can be made.
Those analogues that cause pronounced absorption chịanges also serve as
substrates for the enzyme. The two analogues that cause little or no change
are not substrates. The pyridine mucleotides that cause the formation of both
the 450 and 540 me bands are substrates for the peroxd.dase activity of the
enzyme and also for a very weak oxidase activity. The two substrates (NADPH
and NMNH) that cause the production of only the 450 m Band are substrates for
the weak oxidase activity, but are very poor peroxidase substrates.
Change in structure of NADH may affect the turnover of the enzyme and the
dissociation constant of the binary complex / between flavoprotein and pyridine
carboxamide
much tide) to different extents. For example, replacement of the amino group
Cal Vimax) "
of NADH with -H causes a 70 fold decrease in turnover, but only a 5 fold
increase in the dissociation constant of the binary complox. Although the
turnover is low, tłu analogue (reduced pyridinealdehyde adenine dinucleotide)
18 essentially a peroxidase substrate, as shown by the ratio of peroxidase to
oxidase activity. This is in keeping with the hypothesis that the analogues
that elocit both the 450 and 540 m bands are peroxidase substrates. If the
adaning anino group of NAD 18 replaced with -OH, the turnover of the enzyme in the
presence of the resultant analogue (reduced nicotinamie hypoxanthine dinucleotide)
decreases approximately 50 %, whereas the dissociation constant of the binary
complex increases 25-fold. Therefore, replacement of the carboxamide amino
group with -H affects princippally the turnover of the enzyme; replacement of
the adenine amino group with -OH affects mainly the dissociation constant of".
.. the binary complex. : Replacement of both the carboxamide amino 'group
with -H and the adenine amono group with- OH had a greater effect than
either change singly, since reduced pyridine aldehyde hypoxanthine dinucleotide
forms only weak complexes with the enzyme and has virtually no activity as a
-
-
-
-
-
-
-
t
substrate. A carboxamide amino group is not essential, however, since
-
-
-
-
reduced acetylpyridine adenine dinucleotide compares favorably with NADH .
Amadla
with respect to the properties listed in Table
Reduced N-methyl nicotinamie bleaches the flavoprotein, without forming
a visible complex. Neither reduced N-methyl nicotinamide nor reduced NAD-acetone
are substrates for the enzyme. The data in Table emphasize once more the
close similarity in behavior of NADPH and NMNH.
The data presented above suggest that the spectral intermediates are
kinetically active enzyme-substrate complexes. Further support for this idea
alculated from
comes from comparion of the Michaelis constants (K.), der vart van het ce
double reciprocal plati?
Studios, with the dissociation constants (K) of the binary complexes. The
latter were calculated from titration data (Fig. ). It was assumed that
the absorbance at 450 mu is directly proportional to the concentration of the
binary complex and that i mole of NADH is bound per mole of enzyme-bound FAD.
The graphical method of Klotzt indicates that this stoichiometry is
true for NADH, however the method was inapplicable to the other mucleotides
because of their high dissociation constants.
Comparison of K and K shows that over a wide range of values, the ratio
K/K, remains faitly constant. According to the rate equations of Ingraham
and Makower t y , correspondence between the two constants might be expected
ir the mechanism of NADH peroxidation involved the formation of a ternary
complex between flavoprotein, reduced pyridine nucleotide and H2020 followed
by direct dissociation of the complex into products. At high peroxide
concentrationis, tue Ks for reduced payridhe releotide Kook met Hyy
llam apirimallotiinun minimhion powinni en Llimnolentin e molto...tud..
The graphical mi
of Klotz formy
indicates them
this stoichiometry is
true for NADH, however the methoc! was inapplicable to the other nucleotides
because of their high dissociation constants.
Comparison of K and K shows that over a wide range of values, the ratio
K/X, remains fairly constant. According to the rate equations of Ingraham
and Makower t , correspondence between the two constants might be expected
if the mechanism of NADH peroxidation involved the formation of a ternary
complex between flavoprotein, reduced pyridine nucleotide and H202, followed
by direct dissociation of the complex into products. At high peroxide
concentrations, the K, for reduced pyridine rucleotide would be given by
the Briggs-Haldane relation if either (a) there were a random binding
... sequence or (b) there were an ordered binding sequence and H,O, combined
with the enzyme first. To explain the parallelism between K and K, on this
basis, one would further have to assume that the dissociation of NADH from
the ternary complex is approximately the same as that of NA DŘ from the
binary complex, the latter presumably being in equilibrium with free reduced
favoprotein. The differences in conditions between the kinetic and titration
experiments might explain why X is larger than Kg, whereas, according to the
Briggs-Haldane relation, it should be smaller. A much more rigorous treatment
would be required in order to establish the validity of these arguments.“
Investigation of the steady state kinetics has been hampered by the fact
that at low peroxide concentrations, the enzyme is inhibited by NADH.
Present data, obtained with high concentrations of peroxide and other oxidants,
formation
are consistent with the action of a ternary complex, since at high
reductant and oxidat concentrations, the velocity becomes independent of
the concentration of both substrates. ...,
.
i
DISCUSSION
The stable spectrum obtained on addition of excess NADH to NADH
is
....
i
mond
o
.
.----
-
----..........
peroxidaseris very similar to the spectra found on addition of substrate to
acyl-CoA dehydrogenase + 4. Beinert suggested that the stable long wavelength
band of the substrate-treated acyl-CoA dehydrogenase is that of a flavin .
semiquinone. This suggestion was based on the resemblance of the long
wavelength band to the transient band observed on reoxddation of reduced
.
EMF t Beinert observed similar transient bands on hydrosulfite reduction
of acyl-CoA dehydrogenase and on substrate reduction of L-amino acid oxidase.??
Since that time, the typical picture consisting of partial bleaching at 450 me
and formation of a band at about 520 to 600 m, has been described for several
:.. substrate-reduced flavoproteins. 18-21 . .
.. In some cases, the bands habeonly a transient
exdstence f ay, and are not stable in the presence of excess substrate.
The spectral intermediate in the NADH peroddase system seems to be unique in
that it is not only stable in the presence of excess substrate, but also in
the presence of substrate plus hydrosulfite. It was pointed out't that
oxidered
the spectral intermediate formed on addition of NADH to the peroxidase could
not be that of a simple gemiquinone, since the spectrum is modified by the
structure of the substrate. for a simple Semiquinone, che tend the
that the partial bleaching at 450 m Afstaporttiat oddetto his changemohould
be obligatorily accompanied by the formation of the semiquinone band at 540 mi.
The data of Fig. I show that with NADH peroxidase, the 450 and 540 ml bands can be
dissociated from each other by choosing the appropriate substrate. Whatever
the reduction state of the flavin maliyetine be, the results show that there -s mulhe
interaction between the pyridine nucleotide and the flavin, that 18', the
Intermediate 18 a mlavoprotein-substrate complex.,
Two hypotheses were suggested to account for the structure of the visible .
complex between substrate and NADH peroxidase 3:7,9 These were, (a) the complex
between the pyridine nucleotide and FAD might exist in one of several partly
reduced states ( 1e, 1/2 or 3/4 reduced) or (b) the complex might consist of
fully reduced pyridine nucleotide and fully reduced flavin. The latter view
· was favored, because of the experimental evidence that the characteristic spectrum
is obtained in the presence of NADH and hydrosulfite. For example, the
saturation curve shown in Fig. : implies that the complex is in equilibrium
with fully reduced flavoprotein. If the complex were in equilibrium with oxidized
flavoprotein, complete dissociation would be expected in the presence of
hydrosulfite. Since NAD* and NADH give the same saturation curve (in the presence
of excess hydrosulfite) it appears that the complex contains four reducing
equivalents. The structure, FPH,.NADH would account for such results, but would
abviously be unsatisfactory on other grounds. As pointed out, the results of
Fig. are inconsistertwith a simple semiquinone hypothesis, but it is ..
also difficult to see why a complex consisting of fully reduced flavin and fully
- reduced NAD would give rise to the 450 and 540 m absorption bands.
greduced
21
Furthermore, there is reason to believe that at least one of the complexes
formed netween the oxidized NADH peroxidase and reduced pyridine nucleotide
18 not fully reduced. It will be recalled that the spectrum of the complex ·
(fea I)
formed between enzyme and pyridinealdehyde hypoxanthine dinucleotide can be
almost completely discharged in the presence of hydrosulfite. This result
would not have been expected if the complex had been fully reduced to start
with.
In recent years there has been mounting evidence that long wave length :
bands similar, but not identical, to the 540 m band of NADH peroxidase
can be ascribed to a half-reduced form of flavin hat. Particular mention
might be made of the experiments dealing with anaerobic titration of
2poyl dehydrogenase' and NADPH-cytochrome e reductase with
substrate. It might be well, therefore, to reemphasize at this time
a previously-made suggestion which could bring the behavior of NADH peroxidase
into line with that of other flavoproteins. The scheme is shown in Fig. . .
Essentially, it* 18 suggested that there is a third electron acceptor (x)
at the active site. FAD, but not X is reducible by hydrosulfite, to form
the enzyme species II. Following addition of NADH to this reduced enzyme,
. a redistribution of the four reducing equivalents may take place. One such
redistribution is illustrated by you that is the presence of NADH at its
binding site makes possible an electron transfer from FADH, to X, allowing
the flavin to assume a semiquinoid state or some undefined half-reduced state.
The spectrum of W would be partially dependent upon interaction between the
flavin and the pyridine moleotide. The enzyme species JV would not be reducible
' with hydrosulfite, except perhaps if the pyridine nucleotide . binding site were
occupied by a weakly bound substrate.
:
IS
beina
. In more general terms, NADH may induce an alteration in the enzyme, perhaps
a conformation change, which permits interaction between FADH, and some enzyme-
bound electron acceptor.
Since the addition of either NADT or NADH to hydrosulfite-reduced
enzyme produces virtually the same spectral intermediate, it may be assumed
that under the conditions of the experiment, Nad* is rapidly reduced to NADH.
If I accurately represents the reduction state of the enzyme under physiological
conditions, two moles of NADH would be required to produce the kinetically
active enzyme species. Prendere potestat conditions for the absence
would result in
of_daydrocuifstotnly 1 mole of NADH 18e added to oxddized enzyme, the
... the pyridine nucleotide binding site wordt be occupied by NAD*. It would
therefore be necessary to postulate that the same spectrum is obtained
whether the pyridine nucleotide binding site is occupied by oxidized or
reduced NAD. This supposition seems rather unlikely.
The scheme presented above is highly conjectural. It is offered
merely as one possible interpretation of the results. n order to destace
the oxidation stato or favor to the substrate reduced perokidase, it wit *
bo-negossary to prepare sufficient amounts of enzyma to allow anaerobic.
:. -strations of oxidized and chemicality reduced távoprotoint . :::
Determination of the reduction state of the complex will require the ::
preparation of large amounts of enzyme, so that anaerobic titrations of
oxdidized and chemically reduced flavoprotkin with substrate can be carried out.
. ergue
Because of the high degree of purification required, thes approach has not adyet
boen practical. .. !.


iman practical. .
..
"
.
.
.
.....
::::
.
N
...
.
...
.
..
.
..
..
.
.
16
...
Although the studies on NADH peroxidase tot did not define the
oxidation-reduction state of the spectral intermediate, they did demonstrate
that the intermediate has the characteristics of an enzyme-substrate complex.
Spectral evidence for interaction between bound substrate and substrate-
reduced flavoprotein subsequently obtained in other systems to
Massey f y has shown that the shape of the transient lorg wave length
- band formed on reduction of Deamino acid oxidase by substrate varies with the
structure of the substrate used. This enzyme-substrate complex ( in which
the flavin and amino acid were considered to be in radical states) appears
to be involved in the rate limiting step of the catalysis. Yagi and
Ozawa have been able to crystallized the enzyme-substrate complex of D-amino
acid oxidase and D-alanine. With another flavoprotein, lipoyl dehydrogenase,
Veeger and Massey 4 have shown that the semiquinone spectrum is considerably
: modified in the presence of NAD*. 6
...
(The Nap*dependent intermediate 18 in equilibrium with the semiquinone and may be
a catalytically important species? .
It has been mentioned several times that NADH peroxddase is inhibited by
NADH in the absence of H2O2. The spectral Intermediate discussed in this
paper is not identical with the inhibited enzyme species. It has been shown
· that at the high enzyme concentrations used to demonstrate complex formation,
· the flavoprotein is not inhibitod by NADH."
mamap.
org
The fact that the peroxidase forms a visible enzyme-substrate complex
has made it possible to identify some of the substrate groups that are
Involved in binding the substrate to the enzyme. Evidence that the amino
groups of the carboxamide and adenine moieties of NAD* are important as
binding sites has been presented (Fig.1,2 · Table , I, I ) and briefly
discussed. Of most interest is the fact that correlations can be made
between the structure of the substrate and the spectral and kinetic properties
of the enzyme-substrate complex. Formation of both the 450 and 540 m bands is
caused only by pyridine dinucleotides. · Compounds that elicit this characteristio
spectrum are peroxidase substrates and are also substrates for the very weak
oxidase activity of the enzyme. Compounds that cause the formation only
of the 450 m band (NMNH and NADPH) are substrates for the weak oxddase
activity, but are very poor peroxidase substrates. NMNH and NADPH behave
in an almost identical manner as complexing agents and as substrates. This
correspondence in behavior would be expected if the ester phosphate of
NADPH prevented the binding of the adenylic acid moiety. These results
imply that the 450 m band is characteristic of complexes in which only
the AMN moiety of the substrate is bound to the enzyme. Suatu bindingen
.. requires the provence of at least one fond phosphates pour pren
Judging from the results obtained with free NM, the terminal phosphate of
the ribosyl moiety may be involved in binding the NMN portion of a pyridine
dirucleotide to the enzyme. On removal of the 50 phosphate of NMN, the compound
loses most of its ability to form the 450 m band.
These qualitative observations, which suppresset suggest that the visible
- that is found
complexe are kinetically ætive are supported by the parallelism between the
dissociation constants of the visible complex and the corresponding Michaelis
constants. In essence, the results show that only those complexes in which
both halves of a pyridine weitere are enzyme-bound are able to react
efficiently with a H2Oz:
.
d
Summary
The spectral intermediate that is formed on addition of NADH to
NADH peroxidase has the characteristics of an enzyme-substrate complex.
· The spectral change darempany thing that accompanies complex formation
consists of partial bleaching of the 450 m band and formation of a
broad absorption band centered at about 540 m. Thisharacteristic 2-
banded spectrum is characteristically produced by pyridine dinucleotides
· that are peroxddase substrates. Those pyridine dinucleotide analogues that
are not substrates do not elicit the typical absorption changes.
. . Two pyridine nucleotides (NMVHand NADPH) cause the formation only of the
450 m band. These compounds are poor peroxidase substrates, but they
share with active peroxidase substrates the ability to serve as electron
donors for the very weak oddase activity of the enzyme.
manece
· For those pyridine nucleotides which have measurable enzyme activity,
there is a come good correspondence between the dissociation constant
of the binary enzyme-substrate complex and the Michaelis constant derived
from kinetic studies. These qualitative and quantitative correlations
suggest that the complex 18 a catalytically active species. The investigation
: has not provided unequivocal evidence for the reductiun state of the complex.
li
References

in c. gunsalus &R.Y. Stanier, The Bacteria, Vol. II
1. Dolin, M. I., Aron Bitireno Biophy sample of 79551 9
.
academic Press, drejnew Youl, 1961, p.425
2. Dolin, M. I., Arch. Biochem. biophys., 55 (1955) 425.
3. Dolin, M. I., J. Biol. Chem., 225 (1957) 557.
:46 'Dolin, M. I., Blochim. Biophys. Acta., 42 (1960) 61.
5. Dolin, M. I., J. Bacteriol., 77 (1959) 383, 393.
6. Hoskins, D. D., Whiøtelj, H. R. and Mackler, B., J. Biol. Chem. 23? (1962)
* 2647.
7. Dolin, M. I., J. Biol. Chem. 235 (1960) 544.
8.: Dolin, M. I. and Baum, R. H., Bacteriol. Proc., (1965). 96.
9. Dolin, M. I., Arch. Biochem. Biophys., 60 (1956) 499,
10. Burton, R. M., San Pietro, A. and Kaplan, N. O., Arch. Biochem. Biophys.,
70 (1957) 87.
::. ii. Anderson, A. Go and · Berkelhammer, J. Am. Chem. Soc., 80 (1958) 992. :
| 12. Lineweaver, H. and Ark, D., J. Am. Chem. Soc. 56 (1934) 658.
13. Klotz, I. M., Arch. Biochem., 9 (1946) 109.
14. Ingraham, L. L. and Makower, B., J. Phys. Chem., 58 (1954) 266.
:::..15. Beinert, H. and Crane, F. L., in W. D. McElroy and H. B. Glass filteret,
À symposium on inorganic nitrogen metabolism, Johns Hopkins Press, Baltimore,
: 1956, p. 601.
. . 16. Beinert, H., J. Biol. Chem, 225 (1957) 65. :
17. Beinert, H., J. Am. Chem. Soc., 78 (1956) 5323.
18. Massey, V, and Veeger, C., Biochim. Biophys. Acta, 48 (1961)33.
19. Friedmann, A. C. and Vennesland, B., J. Biol. Chem., 233 (1958) 1398.
20. Massey, V... and 01 bson, Q. H., Fod. Proc. 23 (1964) 18. :..
:
... 21. Masters, B. S. s., Kamin, H. Gibson, Q. H. and Williams, C. H., J.
Biol. Chem. 240 (1965) 921.
22. Yagi, K. and Ozawa, T., Biochim. Biophys. Acta, 67 (1963) 685.
23. Veeger, C. and Massey, V., Biochim. Biophys. Acta, 67 (1963) 679.
.
...
i.
.
.
...
.
.
.
.
.
.
....
. . TABLE I
Maximum Intensities_of_450- and 540-mu bands: hydrosulfite-reduced
NADH...peroxidase plus pyridine nucleotides

450mmu band
540-mu band
A
T
Leon
0.36
0.25
1,95
.3
L
1.70
2.2
1.95
5.8
1.45
1.20
4.8
1.33
1. Enzyme (oxidized)
2. Enzyme + bydrosulfite
3. (2) + NADT or NADH
4. (2) + APYAD*
+ NADA
6. (a) + PYAAD
(2) * PYARD
8. (2) + NADP* 1
. 9. (2) + NN
10. (2) + ribosyi nicotinamide
21. (2) é reduced. NAD-acetone
12. (2) + primary acid modification
product NADH** .
1.087
0.26
.1.3
0.51
5.8
0.25*
o
.
6.0
0.257
o
.
: ......
......
0.256
o
-0.20
0.254
o
2.2
0.10
0.256
o
.
..
.
..
..
..
.
.....
.
...
memo
mi.
Conditions 2 Tor Mig 2, with saturating concentrations of pyridine nucleotides.
A = Extinction for complex minus extinction for bydrosulfite-reduced enzyme, in my
* APYAD* : toutes causes large increase in absorbance at 450 mu when mixed with
hydrosulfite; results are therefore not presented.
**Presumably the trlhydromonohydroxy nicotinamias analogue of NAD (11).
twith this analogue, maximm 18 shifted to 510 says and absorbance 18 approximately
20 per cent higher than at 540 m.
.
Banif 18 within a few per cent of that found for Inc 2. Difference to considered
ummisnot67).
TABLE II
Comparison of reduced pyridine nucleotide analogues as
substrates for NADH_peroxidase

Relativet
· Intensity
.
Peroxidase
activity
(relative
velocity)
:
in
Substrate :
.. .
..
450-304 540-TALE
.
bana
baria
*
NADH
*****
APYADH
NADH
M". KM
x 10-6 1.6 x 10-5 12.7
x 10-6 5.6 x
204 4.2 X
10-5 8.3 %
PYAADH
PyAHDH
NADPH
5.0 x 100:97 x 103 1:9
10-3 4.6
NMNH
0:20
N-Methyl nicotinamide.
099
Standard assay conditions, på 6,5, temperature 24°C. Oxidrse reactions were measured
in oxygen-saturated solution.
*Rates for 1024 reductant, 1,0, as acceptor. For Vase multiply the relative velocities
by the following factors: NADH, 1.03 | APYADH 0.915€ NHDH, 2.6;...
PYAADH, 1.2.
*From 15 m in hydrosulfite-reduced system (Table 1).
#applies to oxidase activity. Peroxidase activity with these substrates follows :.....
first-order Kinetiče::with rospect: to bydrogen.donor. · ·
Table II (cont.)
4 Relative velocity with oxygen as acceptor 18 0.015. This rate 18 not increased in the
presence of peroxide.
VO/V. - ratio of peroxidase to oxidase activity, with 20** M hydrogen donor.
K - Michaolle constant, derived from kinetic studies.
K - Dissociation constant determined from studies on complex formation (see Fig. 2).
i.
.
From
From (7)..
(2).
.
..
.
..
...
.
903.5
-
-
--
SPECTRA OF THE NADH PEROXIDAŞE - SUBSTRATE COMPLEXES
• OXIDIZED ENZYME
+ NADH-L
+ NADPH-
+ PyAHDH
+ HYDROSULFITE

-
oc-----
ABSORBANCE
como-o-o-o-o-o-
-D-DDAD
o
-Ams=========
-oo-ee
.
380
.420
460 ' 500 -
WAVE LENGTH (my)
540
'
580
620
join
fig. 1.
nel
----
-----------------
9036
TITRATION OF HYDROSULFITE-REDUCED NADH PEROXIDASE
WITH PYRIDINE NUCLEOTIDES
.
• NAD+
O NADH
o? PyAHD*
_
D NHD*
ANADPH
A NMN*
RIBOSYL NICOTINAMIDE
0.507

A ABSORBANCE AT 450 my
0.8 1.2 1.
6 2 .0 2.4
PYRIDINE NUCLEOTIDE ANALOGUE (umoles/ml)
+
-
,
Fig. 3
:
I
som
i
habang
NADH
.
.
o
NADH
FADH
FLORA
...
.
-
---
-
-
.
T
*
.
..
.
Legenda. Lor. figures
MADH
. ."Fig. 1: Spectrum OT W H peroxidase in the presence of various reduced
pyridine nucleotides. ,"Bozyme, 0.1 ml. 1n"0.95 M potassium phosphate,
NADH
på 6.5 (equivalent to 0.071 umole bound FAD por mi.); o, enzyme .plus Dumat)
NADPH
2.0 wnoles per ml.; 0, enzyme plus N, 2.5 woles per ml.s broken line,
enzyme plus -pyridinealdehyde mercantling dirucleotide
Inealdehyde att SDPH, 1.2. umoles per mi.; A, enzyme plus
bydrosutrite, 5 ml. or 20 per cent Bodium hydrosulfite la 10 per cent Naucoz.
Results corrected for any light absorption by reagents and Tor dilution. Froml?).
"718. 2.:"Titration or biyārosulfite-reduced enzyme with pyridine pucleotides.
(2)
"Conditions:described in the lettous section. Brazyme equivalent to :0.073 umole,
of bound CAD per m2... NADTO, NAOH n o n authene dinelistide
of bound PAD per mi..., DAS 0,
, pyridinealdehyderabadon DPH
0, doamtoo-Army a, eles, santo, skelet trodde, mibanye micitiramide

1

(mestiramide-hypoxanthine denivelestide.)
Fig. 3. Suggested structures for the intermediates formed between
NADH peroddase and NADH in the presence of hydrosulfite.
.
is
Cris
Footnotes
m
in
warna med
The following abbreviations are used for the oxidized and reduced forms
respectively of the pyridine nucleotide analogues: 3-acetyl pyridine adenine
dinucleoti.de, APYAD, APyADH; 3-pyridinealdehyde adenine. dinucleotide, PYAAD*,
PyAADH: nicotinamide hypoxanthina dinucleotide, NHD*, AHDA; 3-pyridinealdehyde
hypoxanthine dinucleotidó. PyAHD, PYAHDH.
E
r
..
.?
-
-
-
:o
.
.::.
.
''';
.;;
i
.

1
1
my phone more than one time there are
DATE FILMED
16 / 29 /65
revisornica
-
propriimament commen
vi
äter
mm
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a
moj
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....
....
.