Agricultural and Biological Chemistry

ISSN: 0002-1369 (Print) (Online) Journal homepage: http://www.tandfonline.com/loi/tbbb19

Ester Formation by Alcohol Acetyltransferase from

Brewers’ Yeast

Kazuo Yoshioka & Naoki Hashimoto

To cite this article: Kazuo Yoshioka & Naoki Hashimoto (1981) Ester Formation by Alcohol
Acetyltransferase from Brewers’ Yeast, Agricultural and Biological Chemistry, 45:10, 2183-2190

To link to this article: http://dx.doi.org/10.1080/00021369.1981.10864861

Published online: 09 Sep 2014.

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 Agric. Bioi. Chern., 45 (10), 2183 ",,2190, 1981 2183

Ester Formation by Alcohol Acetyltransferase

from Brewers' Yeast

Kazuo YOSHIOKA and Naoki HASHIMOTO

The Research Laboratories of Kirin Brewery Co.,
Miyahara, Takasaki 370-12, Japan
Received November 18, 1980

Alcohol acetyltransferase responsible for the formation of acetate esters during beer fermen-
tation was found to be localized at the cell membrane of brewers' yeast. This cell membrane-bound
Downloaded by [Universidade de Sao Paulo] at 09:21 09 December 2015

enzyme was purified 120-fold by solubilization with Triton X-100, gel filtration on a Sepha-
rose 6B column and chromatography on a DEAE-Sephadex A-50 column. The enzyme was
most active at 30°C at pH 7 '" 8. It was least active against C 3 alcohol among C 1 '" C6 alcohols, and
slightly more active against straight-chain alcohols than against branched-chain alcohols with the
same carbon number. The enzyme was strongly inhibited by unsaturated fatty acids, heavy metal
ions and sulfhydryl reagents.

The accepted theory on the mechanism of
ester formation by brewers' yeast is based
largely on the works of Nordstrom, who show-
ed that ethyl acetate was not formed to any
great extent through esterification of ethanol
with acetic acid, l ) and that several factors
influencing the supply and consumption of
acetyl-CoA affected the formation of ethyl
acetate during fermentation. 2 ,3) The biosyn-
thesis of acetate esters was thus proposed as
occurring through the esterification of al-
cohols with acetyl-CoA, probably in the pres-
ence of an unknown enzyme. Howard and
Anderson supported this proposal with evid-
ence that ethyl acetate was formed from
ethanol and acetyl-CoA by the sediment pre-
pared at 105,000 x 9 from cell-free prepara-
tions of brewers' yeast,4) Ishikawa, Momose
and Yoshizawa suggested that the ester syn-
thesizing enzyme of sake yeast was also as-
sociated with microsomal fractions sediment-
ing at 105,OOOxg. 5 ,6) Yamakawa, Goto and
Yokotsuka succeeded in fractionating the es-
ter synthesizing enzyme from acetone powder
obtained from Cladosporiu:m cladosporioides,7)
affect the flavor quality of alcoholic beverages.
However, there are few reports on the for-
mation of acetate esters by cell-free prepara-
tions of yeast, and little is known about the
localization and the properties of the ester
synthesizing enzyme, which should be. called
alcohol acetyltransferase (acetyl-eoA : alcohol
acetyltransferase). In this paper, the locali-
zation of this new enzyme at the cell mem-
brane of brewers' yeast and the properties of
the partially purified enzyme are reported.
MATERIALS AND METHODS
Yeasts. A typical brewers' yeast (Saccharomyces uva-
rum) was used for the enzyme preparation. Saccharomyces
cerevisiae (NCYC No. 240), a sake yeast (Kyokai No.7), a
wine yeast (IFO 2260) and Hansenula anomala (NI 7572)
were also cultured in a brewers' wort and used for the
experiment on ester formation by. intact cells.
Chemicals. Zymolyase 60,000, a product of Kirin
Brewery Co., Ltd., was used for preparation of the cell
membrane-bound enzyme. CoA derivatives and phospho-
lipids were purchased from Sigma Chemical Co., Ltd.,
USA. All other chemicals were of analytical grade.

but did not establish the localization of the Assay of alcohol acetyltransferase activity. The reaction
enzyme in this microorgap.ism. mixture for assay of the enzyme activity contained 0.8 mM
Ester formation during beer fermentation is of acetyl-CoA, 15 mM of isoamyl alcohol and an appro-
important for brewers because esters greatly priate amount of the enzyme in MilS phosphate buffer or
 2184 K. YOSHIOKA and N. HASHIMOTO
M/10 Tris-HCI buffer, pH 7.5. In a 10-ml reaction flask
with a silicone rubber stopper, 1.5 ml of the reaction
mixture was incubated for 60 min at· 25°C. When the
activity of 10 mg of cell membrane-bound enzyme, 100 mg
of the supernatant fraction, or 100 mg of centrifuged yeast
was assayed, the reaction mixture was incubated with
gentle shaking. Enzyme action was terminated by the
addition of I g of sodium chloride; next, 0.4 ,umol of n-
buthanol was added to each flask as the internal standard.
The flask was heated at 40°C for 20",-, 30 min and isoamyl
acetate content was determined by head space gas chro-
matography under the following conditions.
Gas chromatograph: Hitachi K-53 gas chromatograph
with dual flame ionization detectors
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Column: Stainless-steel column, 2 m x 3 mm i.d., pack-
ed with 10% PEG 1540 on Diasolid L
Column temperature: 90°C
Injection temperature: 160°C
Sample size: Head space gas, 1 ml
Carrier gas: N 2 , 50ml/min
Hydrogen flow rate: 40 ml/min
Air flow rate: 1000 ml/min
One unit of alcohol acetyltransferase was defined as the
amount of the enzyme that produced 1,umol of isoamyl
acetate per min.
Ester formation from alcohols other than isoamyl
alcohol by the action of alcohol acetyltransferase. was
assayed with 0.8 mM of acetyl-CoA and 15 mM of the
alcohols. Inhibition of the enzyme was determined in M/10
Tris-HCI buffer, pH 7.5, with 0.16",-,0.18mU of the
purified enzyme.
Assay of the reverse reaction of esterases. The formation
of ethyl acetate and isoamyl acetate from ethanol and
isoamyl alcohol, respectively, by the reverse reaction of
esterases was assayed in 1.5 ml of Mil 0 acetate buffer, pH
5.0, containing 15 mM of each alcohol, 7 mM of magnesium
chloride and 100 mg of centrifuged yeast. The reaction
TABLE 1. FORMATION OF ACETATE ESTERS BY INTACT CELLS
The formation of acetate esters was assayed with 100 mg of each centrifuged yeast according to the method
for assay of alcohol acetyltransferase activity or the reverse reaction of esterases. All figures are indicated as the
amount per 10 g of centrifuged yeast.
Acetate
esters Substrate
formed S. uvarum
Isoamyl Isoamyl alcohol, acetyl-CoA
acetate
(ppm) Isoamyl alcohol, acetic acid
Ethyl Ethanol, acetyl-CoA
acetate
(ppm) Ethanol, acetic acid
mixture was incubated with gentle shaking for 60 min at
25°C in a 10-ml reaction flask with a silicone rubber
stopper. Ethyl acetate or isoamyl acetate content was
determined by head space gas chromatography under the
same conditions as those for assay of alcohol acetyl-
transferase activity.
Determination ofprotein content. The protein content of
the enzyme solution was determined by the modified
Lowry method8 ) with bovine albumin as the standard.
Sodium dodesyl sulfate was added to the reaction mixture
to prevent the formation of a precipitate due to the
presence of Triton X-100. The protein content of the cell
membrane-bound enzyme was calculated by· multiplying
its total nitrogen value determined by the Kjeldahl meth-
od, by 6.25.
RESULTS AND DISCUSSION
The role of alcohol acetyltransferase in the
formation of acetate esters by brewers' yeast
In order to elucidate the role of alcohol
acetyltransferase or esterases in the formation
of ethyl acetate and isoamyl acetate, the most
popular esters found in alcoholic beverages,
esters formed by lOOmg of the centrifuged
yeasts were' determined.
As shown in Table I, brewers' yeast, as well
as sake yeast and wine yeast, produced iso-
amyl acetate from isoamyl alcohol only in the
presence of acetyl-CoA. In contrast, these
yeasts produced ethyl 'acetate from ethanol
and .acetic acid as well as from ethanol and
acetyl-CoA. This suggests that the formation
of isoamyl' acetate depends exclusively on the
action of alcohol acetyltransferase, whereas
Brewers' yeast
Sake Wine Hansenula
yeast yeast anomala
S. cerevisiae
78 38 81 42 2
0 0 0 0 27
13 9 9 5 191
7 10 6 9 396
 Ester Formation by Alcohol Acetyltransferase
the formation of ethyl acetate depends on both
alcohol acetyltransferase and the reverse re-
action of esterases. Schermers, Duffs and
MacLeod have observed the formation of
ethyl acetate from ethanol and acetic acid by
the reverse reaction of an esterase of brewers'
yeast. 9 )
The formation of acetate esters by
Hansenula anomala was unusual compared
with that of other yeasts examined. Hansenula
anomala produced isoamyl, acetate mainly
from isoamyl alcohol and acetic acid,but
produced only small qua~ntities from isoamyl
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alcohol and acetyl-CoA. This yeast also pro-
duced a very large amount of ethyl acetate
from ethanol, especially in the presenc~ of
acetic acid. This is contrary to reports that
esterases, including Hansenula anomala, 10'" 13)
were not involved in ester formation by yeast.
The formation of esters by this yeast seems to
depend primarily on the reverse reaction of
esterases.
Preparation of cell membrane-bound enzyme
and localization of alcohol acetyltransjerase
activity
To prepare cell membrane-bound enzyme
not containing intact cells, brewers' yeast 'cells
were treated with Zymolyase three times in
M/15 phosphate buffer, pH 7.5 at 25°C with
gentle shaking. After each treatment, the pre-
cipitate was separated from the supernatant by
centrifugation at 5500xg for lOmin. At the
first lysis, 20 g of the centrifuged yeast was
treated with 30 mg of Zymolyase for 2.5 hr in
400 ml of the buffer. The precipitate collected
was further treated with 15 mg of Zymolyase
for 2.5 hr in 200 ml of the buffer. After the
second lysis, the precipitate was collected and
treated again with 15 mg of Zymolyasefor
2.5 hr in 200 ml of the buffer. The precipitate
fromthe third lysis was washed with 100 ml of
deionized water, collected by centrifugation at
5500 x 9 for 10min and lyophilized to obtain
cell membrane fraction. The supernatant from
each lysis was saturated with ammonillm sul-
fate and stirred for 1. hr, at 1°C.The resultant
precipitate was collected by centrifugation at
2185
T ABLE II: LOCALIZATION OF ALCOHOL
ACETYLTRANSFERASE IN
BREWERS' YEAST
Activity of alcohol acetyltransferase in each fraction
prepared from 100 g of the centrifuged yeast.
Fraction Activity (mU)
Precipitate after 3rd lysis
(Cell membrane fraction) 54.0
Supernatant after 1st lysis 21'.1
Supernatant after 2nd lysis 3.1
Supernatant after 3rd lysis 1.2
5500xg for lOmin, dissolved in a small
amount of deionized water and lyophilized for
assay of activity in the supernatant containing
microsomal fractions.
Table II shows the distribution of alcohol
acetyltransferase in the brewers' yeast. About
70% of the activity was observed in the pre-
cipitate obtained by centrifugation at 5500 x 9
for 10 min., This fraction was used for further
purification as the cell membrane-bound en-
zyme. The absence of intact cells in this frac-
tion was confirmed by microscopic exam-
ination and observation of the lack.. of the
ability to ferment' glucose to ethanol. When
this fraction was suspended in M/15 phosphate
buffer, pH 7.5 containing 0.25 M sucrose, ho-
mogenized for 2 min in a waring blender and
centrifuged at 700 x 9 for 10 min,about 80%
of the activity of the homogenate wasobserv-
ed in the'precipitate, and most of the residual
activity was observed in the' precipitate after
centrifugation ofthe supernatant at 10,000 x 9
for 20 min.
These results suggest that alcohol acetyl-
transferase of brewers' yeast is mainly located
at its cell membrane.
Partial purification of alcohol acetyltransferase
The following purification procedures were
carried out at 1°C to minimize inactivation of
the enzyme.
Solubilization, of the enzyme from the cell
membrane. The enzyme was solubilized most
effectively by treatment of 1. 5 g of the cell
membrane fraction obtained from 100 g of the
 2186 K. YOSHIOKA and N. HASHIMOTO

centrifuged yeast with 25 ml of 1% Triton X-

100 in M/15 phosphate buffer, pH 7.5. The
mixture was stirred for 1 hr, and the super-
natant was collected after centrifugation at
10,000 x g for 10 min. The precipitate was
washed with 10 ml of 1%Triton X-100 in MilS
phosphate buffer, pH 7.5, and the supernatant
was collected after centrifugation at 10,000 x g
for 10 min. The supernatants were combined
10 20 30
to obtain 25 ml of the dissolved enzyme. The Fraction number (10 ml/:fraction)
total activity of the enzyme remarkably in- FIG. 1. Gel Filtration of the Enzyme on a Sepharose 6B
creased after solubilization from the cell mem- Column.
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brane as shown in Table III. A part of the 0--0, protein; e---e, activity.
enzyme seems to be bound to the cell mem-
brane in an inactive form. Approximately 109
of Sephadex 0-25 was placed in a beaker 50
+J
.c 40 :;
containing approximately 25 ml of the dissolv- ''';
bI.J
(I,)
ed enzyme for 10 min. The swelled gel was then ~
30

removed by centrifugal filtration at 1500 x g ~o

~ g20
for 10 min. The dissolved enzyme was con- =' ..
uo
Q,;.....;
centrated into 5 ml of the filtrate without
'0 ~
significant loss of activity. ~ -10
Gel filtration on a Sepharose 6B column. A- 1~0 200 250
Elution·volume (ml)
5 ml concentrated sample was applied on a
Sepharose 6B column (60 cm x 2.6 cm i.d.) FIG. 2. Estimation of Molecular Weight of the Enzyme
by Gel Filtration on a Sepharose 6B Column.
equilibrated with MilO Tris-HCl buffer, pH
1, y-globulin (human) M.W. 160,000; 2, alcohol acetyl-
7.5, containing 1% Triton X-100. The column
transferase; 3, catalase (bovine) M.W. 240,000; 4, ferritin
was eluted with the same buffer at a flow rate (horse spleen) M.W. 364,000; 5, apo-ferritin (horse) M.W.
of 20 mllhr, and fractions of 10 ml were col- 480,000.
lected. Fractions No. 21 to 24 shown in Fig. 1
were combined for further purification by
chromatography on a DEAE-Sephadex A-50 ,.--------10.05
column. The specific activity of the enzyme o
decreased slightly when the enzyme was frac- 6 ~
'I
"
~
tionated by gel filtration overnight, as seen in 5 ," 1
"
I I
Table III, probably because of the instability ~ bI) , ,
I I

-5-54 : I
of the enzyme solubilized. The molecular >.
.+oJ ~ ,
I '
~
I I
.~.~ 3
weight of the enzyme was estimated to be 'r-! .+oJ
.+oJ 0
! ~
about 220,000 from the elution volume of the I \

marker proteins and the enzyme, as shown in
Fig. 2.
Chromatography on a DEAE-Sephadex A-
50 column. Forty milliliters of enzyme solution
obtained by gel filtration on a Sepharose 6B
column were applied on a DEAE-SephadexA-
50 column (5 cm x 1.5 cm i.d.) equilibrated
with MilO Tris-HCl buffer, pH 7.5 containing
1% Triton X-100. The column was first eluted
~J:2
I \
I \
I \
f \
I \-.~ ...
o 5 10 IS 20
Fraction number (10 ml/fraction)
FIG. 3. Chromatography of the Enzyme Fraction on a
DEAE-Sephadex A-50 Column.
Fractions No. 21,,-,24 shown in Fig. 1 were collected and
subjected to column chromatography.
0-0, protein; e---e, activity.
 Ester Formation by Alcohol Acetyltransferase 2187

TABLE III. PURIFICATION OF ALCOHOL ACETYLTRANSFERASE FROM BREWERS' YEAST

Purification from 100 g of the centrifuged yeast.

Specific
Total Total
activity Purification Yield
Step protein activity
(mU/mg (fold) (%)
(mg) (mU)
protein)

Cell membrane-bound enzyme 864 54.0 0.0625 1 100

Dissolved enzyme 109 88.5 0.812 13 164
Sephar·ose 6B gel filtration 28.6 21.5 0.752 12 40
DEAE-Sephadex A-50
1.26 9.18 7.29 117 17
column chromatography
Downloaded by [Universidade de Sao Paulo] at 09:21 09 December 2015

100 100
,,
,
\
~/
I

\
, 80
I

\
;i
",
\ I

",
~
,/
~60 \
I

>
.~
\
l
,
"
I
-+J I
U I
tU 40
<I)
>
.~
'. ,A'
/

-+J
tU20 ,,/,--

.'
...-l
<I)
0:;
O~ _ _- - - - J l . - -_ _--L-_ _- - - L_ _
0'-------'-------£----10.--.:....----1..
10 20 40 5 6 7 8
Temperature pH

FIG. 4. Effect of Temperature on Alcohol Acetyltrans- FIG. 5. Effect of pH on Alcohol Acetyltransferase

ferase Activity.
The activity at 30°C is shown as 100%.
0--0, partially purified enzyme; e---e, cell membrane-
bound enzyme.
with 100ml of Tris-HCI buffer, pH 7.5 con-
taining 1%Triton X-I 00, and then with 200 ml
of the same buffer containing 1%Triton X-lOO
and M/20 sodium chloride. Fractions No. 16
and 17 shown in Fig. 3 were used as the
partially purified enzyme.
The purification procedure is summarized in
Table III. The enzyme was 120-fold purified
from the cell membrane of brewers' yeast by
solubilization with Triton X-IOO, gel filtration
and column chromatography.
Properties of alcohol acetyltransferase
Effect of pH and temperature. Both the cell
membrane-bound enzyme and the purified en-
zyme were most active at 30°C in a pH range
Activity.
The activity of the partially purified enzyme at pH 7 and
the activity of the cell membrane-bound enzyme at pH 8
are shown as 100%.
0--0, partially purified enzyme; e---e, cell membrane-
bound enzyme.
of 7 ~ 8, as shown in Figs. 4 and 5. The opti-
mum pH of the enzyme was slightly lower-
ed by solubilization of the enzyme from the
cell membrane.
Substrate specificity. The enzyme was most
inactive against C 3 alcohol among C 1 ~ C6
alcohols, and slightly more active against
straight-chain primary alcohols than against
branched-chain alcohols of the same carbon
number, as seen in Fig. 6. The predominance
of ethyl acetate formation during brewing is
not the result of significant alcohol acetyl-
transferase activity against ethanol, but is
instead due to the natural predominance of
 2188 K~ YOSHIOKA and N.· .HASHIMOTO

200
Cell membrane-
TABLE V; EFFECT OF LIPIDS ON ALCOHOL
Partially
purified bound enzyme ACETYLTRANSFERASE ACTIVITY
enzyme
The enzyme activity was determined in the presence of
>-. 150 2 mM of each lipid.
-j-l
.,..,
.,..,:>
-j-l Relative
u Lipid
m 100 activity (%)*
(J.)

.,..,:>
,,:'
, None (Control) 100
-j-l
m
,,
I
r-1 Myristic acid 77
~ 50 I
Palmitic acid 86
...--_.. Stearic acid 86
Oleic acid 36
Linoleic acid 3
o 1 2 4 5 6 I 2 4 6 Linolenic acid 3
Downloaded by [Universidade de Sao Paulo] at 09:21 09 December 2015

Carbon number
Monostearin 42
FIG. 6. Substrate Specificity of the Enzyme for Alcohols. Monoolein 24
Formation of corresponding esters (pmol/min) was as- Steoroyl-CoA 87
sayed with 15 mM of each alcohol and 0.8 mM of acetyl- Palmitoyl-CoA 72
CoA as substrate. The activity for isoamyl alcohol is Oleoyl-CoA 13
shown as 100%. Glycerol 86
0--0, straight-chain primary alcohols; e---e, Tween 40 51
branched-chain alcohols (isopropyl alcohol, isobutyl al- Tween 60 23
cohol, isoamyl alcohol). Tween 80 11
Ergosterol 27
Lecithin 66
TABLE IV. SUBSTRATE SPECIFICITY OF THE Phosphatidylcholine, dipalmitoyl 89
ENZYME FOR BUTYRYL-CoA Phosphatidylcholine, dioleoyl 58
Formation of the corresponding ester (pmol/min) was Phosphatidylethanolamine, dipalmitoyl 91
assayed with 15 mM of alcohol and 0.8 mM of acyl-CoA as Phosphatidylserine 12
substrates. Phosphatidylinositol 3

Relative activity· (%) * The activity without lipid is shown as 100%.

Substrate
Cell membrane- Dissolved Purified
bound enzyme enzyme enzyme
cause it is bound to the cell membrane of
brewers' yeast" with'a part of the enzyme
Isoamyl alcohol
100* 100* 100*
apparently bound in an inactive form.
+ Acetyl-CoA Generally, unsaturated fatty acids in a me-
Ethanol
55 40 31
dium greatly reduce the ester level and satu-
+ Acetyl-CoA rated fatty acids raise it. 14 '" 19) However, the
Ethanol
1.4 1.3 1.3
assumption by Ayrapaa 14 ) that unsaturated
+ Butyryl-CoA fatty acids inhibit the action of the ester
* The activity for isoamyl alcohol and acetyl-CoA is
synthesizing enzyme remains to be
shown as 100%. demonstrated.
Table V shows the effect of fatty acids and
ethanol formation, and due to the partial lipids on the activity of alcoholacetyltrans-
formation of ethyl acetate by the reverse re- ferase purified from brewers' yeast. The en-
action of esterases. The enzyme was identified zyme was strongly inhibited by unsaturated
as alcohol acetyltransferase because of its fatty acids, especially by linoleic acid and
slight tendency to form" ethyl butyrate from linolenic acid. In contrast, saturated fatty
ethanol and butyryl-CoA, as shown in Table acids such as myristic acid, palmitic acid and
IV. stearic ;acid showed· only a slight inhibitory
Effect offatty acids. The effect of lipids on effect. The derivatives of unsaturated fatty
the activity of the enzyme is interesting be- acids such as monoolein, oleoyl-CoA, Tween
 Ester Formation by Alcohol Acetyltransferase 2189

TABLE VI. EFFECT OF METAL IONS ON TABLE VIII. EFFECT OF CYSTEINE ON INHIBI-
ALCOHOL ACETYLYTRANSFERASE TION OF THE ENZYME BY HEAVY METAL
ACTIVITY IONS AND SULFHYDRYL REAGENTS

The enzyme activity was determined by adding I mM of The concentration of each inhibitor in the reaction
each salt to the reaction mixture. mixture is the same with that in Tables V, VI and VII.

Salt' Relative activity (%)* Relative activity (%)

Inhibitor
None (Control) 100 In the absence In the presence
Na·CH 3 COO 101 of cysteine of 10 mM cysteine
KCI 99
K 2 S04 97 None (Control) 100* 56
KN0 3 9r Pb(CH 3 COO)2 o 69
MgCl2 96 CoCl2 17 76
CaS04 95 CuS04 1 6
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SrCl2 103 ZnCl2 3 71

BaCl2 67 AgN0 3 8 43
AICl 3 89 CdCl2 o 84
SnCl 2 76 HgCl 2 2 66
Pb(CH 3 CPOh o PCMB** 2 30
MnCl 2 67 DTNB** 1 64
FeS04 94 Oleic acid 36 30
CoCl 2 17 Linoleic acid 3 3
NiCl 2 32
CUS04 1 * The activity of the control is shown as 100%.
ZnCl 2 3 ** The names of the inhibitors are abbreviated as
AgN0 3 8 shown in Table VII.
CdCl 2 o
HgCl 2 2
inositol. The activity of alcohol acetyltrans-
* The activity assayed without salt is shown as 100%.
ferase bound to the cell membrane seems to be
related to the composition of lipids in the cell
T ABLE VII. EFFECT OF SOME INHIBITORS membrane. Ester formation in relation to li-
ON ALCOHOL ACETYLYTRANSFERASE
pids metabolism is an important subject for
ACTIVITY
future investigation.
The enzyme activity was determined in the presence of Inhibition by heavy metal ions and sulfhydryl
each inhibitor.
reagents. The effect of metal ions on the
Relative enzyme activity is summarized in Table VI.
Inhibitor
activity (%)* None of metal ions examined had a stimu-
latory effect on the activity of this enzyme.
None (Control) 100
Monoindoacetic acid (IAA) 72
Contrary to the observation by Howard and
p-Chloromercuribenzoic acid (PCMB) 2 Anderson that 6/'-18 mM of magnesium ions
5,5'-Dithiobis(2-nitrobenzoic acid) stimulated the formation of ethyl acetate from
(DTNB) ethanol and acetyl-CoA by microsomal frac-
Phenylmethanesulfonyl fluorid (PMSF) 93
2,4,6-Trinitrobenzenesulfotiic acid
tions of brewers' yeast,4) the enzyme separated
(TNBS) 43 from the cell membrane was found to be
somewhat significantly inhibited by 1/'-1 10 mM
* The activity without inhibitor is shown as 100%.
of magnesium ions. The enzyme was strongly
inhibited by heavy metal ions such as lead,
80 and phosphatidylcholine, dioleoyl, had also copper, zinc, cadmium and mercury ions, as
a stronger inhibitory effect on the enzyme than seen in Table VI.
the derivatives of saturated fatty acids. The Table VII shows the effect of some in-
enzyme was also strongly inhibited by ergo- hibitors on the activity of alcohol acetyltrans-
sterol, phosphatidylserine and phosphatidyl- [erase. It was strongly inhibited by sulfhydryl
 2190 K. YOSHIOKA and N. HASHIMOTO
reagents such as p-chloromercuribenzoic acid 4)
and 5,5 /-dithiobis (2-nitrobenzoic acid).
5)
Inhibition of the enzyme was also observed
when it was separated from 5,5 ' -dithiobis (2-
nitrobenzoic acid) by gel filtration on a 6)
Sephadex G-50 column after incubation in the
presence of 2 mM of the reagent for 10 min at
7)
20 o e, while the enzyme eluted from the col-
umn after incuvation in the absence of the 8)
reagent exhibited appreciable activity. As seen
in Table VIII, the strong inhibition of the 9)
enzyme by heavy metal ions except copper
10)
Downloaded by [Universidade de Sao Paulo] at 09:21 09 December 2015
ions or sulfhydryl reagents was diminished in
11)
the presence of 10 mM of cysteine. These re-
sults clearly show that the enzyme is a sulfby- 12)
dryl enzyme.
13)
Acknowledgments. The authors wish to thank the 14)
management of Kirin Brewery Co., Ltd. for permission to
publish this work. They are also grateful for the con- 15)
tinuous encouragement of Mr. T. Itoga, Managing
Director, and Dr. Y. Kuroiwa, Director of the Research 16)
Laboratories.
17)
REFERENCES
18)
1) K. Nordstrom, J. Inst. Brew., 67, 173 (1961).
2) K. Nordstrom, J. Inst. Brew., 68, -398 (1962). 19)
3) K. Nordstrom, J. Inst. Brew., 69, 142 (1963).
D. Howard and R. G. Anderson, J. Inst. Brew., 82,
70 (1976).
T. Ishikawa, H. Momose and K. Yoshizawa,
Abstract of Papers, Annual Meeting of Nippon
Nogeikagaku Kai, Tokyo, April, 1979, p. 408.
T. Ishikawa, H. Momose and K. Yoshizawa,
Abstract of Papers, Annual Meeting of Nippon
Hakkokogaku Kai, Osaka, October, 1979, p. 147.
Y. Yamakawa, S. Goto and I. Yokotsuka, Agric.
Bioi. Chem., 42, 269 (1978).
C. S. Wang and R. L. Smith, Anal. Biochem., 63, 414
(1975).
F. H. Schermers, J. H. Duffs and A. M. MacLeod, J.
Inst.Brew., 82, 170 (1976).
J. L. Peel, Biochem. J., 49, 62 (1951).
J. Tabachnick and M. A. Joslyn, J. Bacteriol., 65, 1
(1953).
J. Tabachnick and M. A. Joslyn, Plant Physiol., 28,
681 (1953).
T. Baba, Hakkokogaku Zasshi, 34, 79 (1956).
T. AydipiHi and I. Lindstrom, 14th, Eur. Brew.
Conv., Proc. Congr., Salzburg, 1973, p. 271.
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