522 AROMATIC AMINO ACIDS [65]

weight of about 100,000 is suggested) The enzyme contains 2 g-atoms

of iron per mole of enzyme. The iron is found to be in the ferric state. 6
A solution of the purified enzyme shows an absorption spectrum with
a broad peak between 400 and 600 mtz and a shoulder at 322 mlz;
A 440
I ~ = 0.519; az•l280
~ = 8.93.

eT. Nakazawa, Y. Kojima, H. Fujisawa, M. Nozaki, O. Hayaishi, and T. Yamano, J. Biol.

Chem. 240, PC3225 (1965).

[65] Metapyrocatechase (Pseudomonas) a

By MITSUHIRO NOZAKI

H
I
,- o. .c=o o
H H

Catechol a-Hydroxymuconic
• -semialdehyde

Assay Method
Principle. The enzyme activity is assayed by measuring the increase
in optical density at 375 mtz due to the formation of the reaction
product, a-hydroxymuconic ¢-semialdehyde. laa Alternatively, the ac-
tivity may be assayed polarographically by measuring the oxygen
consumption?

Reagents
Potassium phosphate buffer, 0.5 M, pH 7.5
Catechol, 0.01 M. Dissolve 11 mg ofcatechol in 10 ml of water
Enzyme. Dilute enzyme with cold buffer containing 10% acetone
to obtain a solution containing 0.5-1.0 unit/ml.

Procedure. 2"4 The assay system contains in a final volume of 3.0 ml:
0.1 ml of catechol, 0.3 ml of potassium phosphate buffer (pH 7.5), and

tEC 1.13.1.2; catechol : oxygen 2,3-oxidoreductase; catechol 2,3-oxygenase.

taS. Dagley and D. A. Stopher, Biochem.J. 73, 16P (1959).
~Y. Kojima, N. Itada, and O. Hayaishi,J. Biol. Chem. 236, 2223 ( 1961).
SB. Hagihara, Biochim. Biophys.Acta 46, 134 ( 1961 ).
4M. Nozaki, H. Kagamiyama, and O. Hayaishi, Biochem. Z. 338, 582 (1963).
[65] METAPYROCATECHASE 523

0.1 ml of enzyme in a cuvette with a 1-cm light path. The reaction is

initiated by the addition of enzyme at 24 °. The rate of increase in ab-
sorbance at 375 m/.~ is followed with a recording spectrophotometer.
Definition of Unit and Specific Activity. One unit of enzyme is defined
as that amount which oxidizes 1 micromole of catechol per minute at
24 °. Since the molar extinction coefficient of the product is approxi-
mately 4.4 × 104 under these conditions, 5 one unit of the enzyme
activity corresponds to an optical density increase of 14.7 per minute.
Specific activity is expressed as units per milligram of protein. Protein
is determined by the method of Lowry et al. 6 using crystalline bovine
serum albumin as a reference protein.
P u r i f i c a t i o n P r o c e d u r e 4a

Growth of Bacteria. Cells of Pseudomonas arvilla (ATCC 23973) are

grown in a medium containing 0.30% (NH4)2HPO4, 0.12% KH2PO4,
0.5% NaC1, 0.02% MgSO4"7H20, 0.05% yeast extract, and 0.3% ben-
zoate for 20 hours at 30 ° with vigorous aeration. Cells are harvested
with the aid of a Sharpies centrifuge and washed once with 0.85% KCI
solution. They are stored in a deep freezer (--20°) with little loss of
activity for several months. All subsequent extraction and purification
procedures are carried out at 3-5 ° .
Step 1. Crude Extract. Forty grams of the wet frozen cells are suspended
in 400 ml of 0.05 M phosphate buffer, p H 7.5, and disrupted by sonic
oscillation at 10 kc for 10 minutes. The supernatant fluid (crude ex-
tract) is separated from the residue by centrifugation at 15,000 g for
10 minutes. Since the presence of a low concentration of an organic
solvent such as acetone or ethanol has been found to protect the enzyme
almost completely from inactivation by air, as described below, both
chromatography and crystallization procedures are carried out in the
presence of 10% acetone.
Step 2. Acetone Fractionation. To the crude extract, 200-300 jzg of de-
oxyribonuclease are added, and the mixture is kept at room tempera-
ture for 15 minutes. One volume of cold acetone is added to the mixture
and the resulting precipitate is removed by centrifugation at 10,000 g
for 10 minutes. A second volume of acetone is added to the supernatant
to give a final concentration of 66% acetone, and the resulting pre-
cipitate is collected by centrifugation and suspended in about 20 ml of
0.05 M phosphate buffer, pH 7.5, containing 10% acetone (hereafter
5This value was experimentally obtained and is much higher than the reported value,
3.3 × 104.2
60. H. Lowry, N. J. Rosebrough, A. L. Farr, and R. J. Randall, J. Biol. Chem. 193, 265
(1951); see also Vol. III [73].
CM. Nozaki, H. Kagamiyama, and O. Hayaishi, Biochem. Biophys. Res. Commun. 1 l, 65 (1963).
524 AROMATIC AMINO ACIDS [65]
referred to as acetone-buffer). The insoluble material is removed by
centrifugation, and the clear supernatant solution is dialyzed against
acetone-buffer overnight at 3-5 ° .
Step 3. Chromatography on DEAE-Cellulose. The dialyzed enzyme is
passed through a DEAE-cellulose column (15 cm x 2 cm diameter)
previously equilibrated with acetone-buffer. After washing the cellulose
column with acetone-buffer, the enzyme is eluted with a linear gradient
established between 500 ml of acetone-buffer (mixing chamber) and
500 ml of acetone-buffer containing 5% ammonium sulfate (reservoir).
The enzyme is eluted at a concentration of ammonium sulfate of
1.5-2.5%.
Step 4. Crystallization. To the combined active fractions (320 ml),
1.7 volumes of cold acetone are added to precipitate the enzyme. The
precipitate is collected by centrifugation at 10,000 g for 10 minutes,
dissolved in a small amount of acetone-buffer (about 5 ml) and dialyzed
against acetone-buffer overnight. Finely powdered ammonium sulfate is
then gradually added to the dialyzed solution until it becomes slightly
turbid. White needlelike crystals appear after several hours at 0 °. Re-
crystallization is carried out by repeating the crystallization process
described above. The yield of enzyme and specific activity at each step
in the procedure are shown in the table.

PURIFICATION OF METAPYROCATECHASE

Volume Specific Yield

Fraction (ml) Units activity (%)
1. Crude extract 530 26,700 4.0 100.0
2. Acetone fraction 20 21,400 20.9 81.0
3. DEAE-cellulose 320 13,700 105.0 51.7
chromatography
4. 1st Crystallization 5 6,000 110.0 22.4
2nd Crystallization 3 5,300 114.0 19.8
3rd Crystallization 3 4,000 116.0 15.0
Supernatant from 3rd 3 500 102.0 2.0
crystallization

Properties
Stability. The enzyme is easily inactivated by various oxidizing agents,
such as air or H202. 2"4 This inactivation appears to be due to oxidation
of ferrous ion to the ferric form. s'° T h e presence of a low concentration
SH. Taniuchi, Y. Kojima, F. Kanetsuna, H. Ochiai, and O. Hayaishi, Biochem. Biophys.
Res. Commun. 8, 97 (1962).
9M. Nozaki, K. Ono, T. Nakazawa, S. Kotani, and O. Hayaishi, J. Biol. Chem. 243, 2682
(1968).
[65] METAPYROCATECHASE 525

of an organic solvent such as acetone or ethanol is found to protect the

enzyme almost completely from inactivation by these oxidizing agents
even at 300.4 The enzyme is most stable at pH 7.5. The crystalline
preparation in the presence of 10% acetone has been stored in a re-
frigerator with no detectable loss of activity for several months.
pH Optimum. The pH optimum for the enzyme activity is 6.5. Molar
extinction coefficient of the product at 375 m/z is approximately 2.3 x 10s
at pH 6.5. TM
Kinetic Constants. The Km values for catechol and oxygen are 3 x 10 -6
M and 7 x 10 -n M, respectively. The molecular activity of the enzyme
is approximately 16,000 moles per minute per 140,000 g under the
standard assay conditions.
Inhibitors. 9 Enzyme activity is inhibited by a variety of nitrogen-
containing aromatic compounds, including o-phenanthroline, a,a'-
dipyridyl, m-phenanthroline, a-naphthoquinoline, quinoline, and
pyridine. Inhibition by these agents is shown to be competitive with
respect to the substrate, catechol. Organic solvents, such as acetone or
ethanol, also inhibit the enzyme activity nearly competitively with
respect to catechol. The enzyme is inactivated by an excess amount of
sulfhydryl inhibitors such as p-chloromercuribenzoate only when they
are preincubated with the enzyme.
Substrate Specificity.TM The enzyme has a broad substrate specificity.
The relative maximum rates are catechol 100, 4-methylcatechol 100,
3-methylcatechol 62, 4-chlorocatechol 51, pyrogallol 33, protocatechu-
aldehyde 20, and protocatechuic acid 0.15. T h e enzyme is, however,
rapidly inactivated during catalysis when these analogs are used as
substrate.
Molecular Properties. The sedimentation constant (s20,w) and diffusion
constant (Dz0,w) of the enzyme are 5.54 × 10-13 (cm/sec), and 3.92 x 10-7
(cm2/sec), respectively. The molecular weight is calculated to be approxi-
mately 140,000, assuming a partial specific volume of 0.75. Depending
on the growth conditions and the amount of iron removed during the
purification procedures, the specific activity of the final purified prep-
aration varies considerably. In the current work, the enzyme with a
specific activity of about 110 micromoles per minute per milligram of
protein contains 1 g-atom of iron per mole of enzyme. 9 However, other
crystalline preparations with a specific activity of about 250 have been
obtained from the same bacteria under different growth conditions
that contain approximately 3 g-atoms of iron per mole of enzyme, tt
1°Unpublished data in our laboratory.
I~M. Nozaki, Y. Kojima, T. Nakazawa, H. Fujisawa, K. Ono, S. Kotani, O. Hayaishi, and
T. Yamano, in "Biological and Chemical Aspects of Oxygenases" (K. Bloch and O.
Hayaishi, eds.), p. 347. Maruzen, Tokyo, 1966.