Reprinted from Journa. or Bactiuto.oay
Vol. €8, No. 3, pp. 265-275, Saptember, 1954
Printed in USA,

_ON THE ROLE OF THE INDUCER IN TILE SYNTHESIS OF MALTASE IN YEAST!

$. SPIEGELMAN anp HARLYN 0. HALVORSON? 3

Department of Racterivlogy, University of Illinois, Urbana, Illineis

Received for publication February 4, 1954

Qne aspect of the process of the induced
dynthesis of enzymes which is relatively amenable
to experimental analysis is the role of the inducer.
It has been established in a varicty of cases that
utilizability is not a necessary attribute of cffec-
tive inducers. Thus, Spiegelman et al. (1947)
demonstrated that. maltose can. induce maltase
at pH values that prechide metabolism of the
maltose by fully adapted yeast cells. Analogues of
natural substrates have been employed for
similar purposes. Thus, a@-methy]l glucoside
ean be shown to be an inducer of maltase at

_cuneentration levels at which utilization cannot

be detected (Spiegelman, 1948). Monod et al.
(1951) have demonstrated similarly that meli-
tnose, though not metabolizable by Kscherichia
cold, is nevertheless an extremely powerful
inducer of B-galactosidase.

Such experiments eliminated interpretations
of inducer function which demand active and
detectable metabolic transformations of the
inductor, such as might be required were indue-
tion to involve the accumulation of unique
intermediates derived from the inducer, in the
sense suggested by Leibowitz and Hestrin
(3915). The data do not exclude mechanisms
which involve the conversion or fixation of a very
small number of inducer molecules.

More pertinent to the purposes of the present
paper, however, is the fact that these experiments
leave open the question. of whether inducers
must. form complexes, with the enzyme being
formed as a sina qua non of induced synthesis of
enzyme.

Mach of the earlier and even some of the more
“reeent, (Mandelstam, 1952; Mandelstam and
Yudkin, 1952) speculations on the role of the
inducer have assumed such complex formation

? This investigation was aided by a grant from
the National Cancer Institute of the U.S. Public
Health Service. :

? Pre-doctoral Fellow of the U.S. Publie Health
Service during period of this investigation.

? Present addfess: Department of Bacteriology, .

Iniversity of Michigan, Ann Arbor, Michigan.

as a necessary concomitant of enzyme formation.
Yudkin (1938) was the first to provide an

‘explicit formulation of this concept, and he did

so in, terms of a mass action hypothesis in which a
substrate by combining with enzyme drives the
reaction toward the conversion of preeursor
into enzyme. -

To explain the kinetics of enzyme synthesis
which cin be observed under. certain conditions,
and certain features of the transmission of
enzyme forming capacity, Spiegelman (1945,
19-46) was Ied to abandon the simple mass action |
theory and to suggest a modilication which
assumed that an autocatalytie clement (the
plasmagene) was involved in’ the synthetic
precess. Subsequently, Monod (1947) proposed
an analogous hypothesis with however more
restrieled genetic autonomy than was inherent
in the plasmagene theory, Poth the Monod and
the Spiegelman’ modifications, nevertheless,
retained the central thesis that the inducer:
functioned by complexing with the enzyme.
The retention of this concept was conditioned not
only by its attractive simplicity, but also because
it suggests an explicit explanation for the
specificity which appeared to characterize the
induction of enzyme synthesis. It possessed the
added advantage of providing a conerete, and in
principle, testable mechanism of at least one
reasonably well defined portion of the inductive
process. .

An uncomplicated application of the complex-
ing coneept leads to several predictions, the
experimental violation of any one of which would
require cither its revision or complete abandon-
ment, We may list some of these predictions as
follows: (1) Substances whieh enn “eamplex”
with enzyme should be effective inductors.
(2) Substances shown to be ineapable of complex
formation with a given enzyme likewise should
he unable to induee this enzyme. (3) The dis-
sociation constant of a substance measured in
terms of its inductive effeet on enzyme synthesis
should be comparable in magnitude to the value
obtained in experiments in Whieh the constant is

265
266 S$. SPIEGELMAN AND HARLYN O, ITALVORSON

derived from the complexant: properties of the
inducer with enzyme,

It should be noted that the term “complex
formation” employed here is meant to subsume
both the specific type (characterized by conbina-
tion between an enzyme and its ‘substrate or a
competitive inhibitor) and the nonspecific type
(characterized by a complex between an enzyme
and. a noncompetitive inhibitur). Thus, proof
that an indueer decsnet ferm a specific “complex”
with enzyme docs not eliminate it as an enzyine

complexant since if leaves open the possibility ,

of. nonspecific complex: formation, “Chere is no
a priort reason for believing that combinations
of the latter type cannot function in the process
of enzyme synthesis. However, violations of.
prediction: (3) above would eliminate the
involvement of either complexant type.

The design of experiments te test the validity
of these predictions is subject to one important
restriction. Thus far, the induction of enzyme
synthesis has been achieved only with intact
cells. ‘The comparison of the hehavior of a
substance “as an inducer and as a complexant

with enzyme must, therefere, be. made on the

intuet cell, Thus, if a substance were shown to

be a complexunt with the enzyine iu an i rita .

test and exhibited no inductive capacity, it could
be argued that it cannot “penetrate” the intact
cell, Analogous or comparable arguments can be
leveled at suecessful violations of any of the
three predictions which are achieved by compar-
ing information obtained from ia vitro experiments
on enzyme with ia rire inductions, The data to
be dixcussed therefore will be confined for the
most part to ir vive comparigans, ,
A clear-cut experimental contradiction of the
complexing concept came from’ Lederberg’s
(1951) examination ‘of mutational effects on the
inductive process. He exhibited a mutant of -E.
Coli (Lacry which could produce 8-gulactosidase
providing alkyl ealactosides were employed as
inducers, but which did not respond to lactose
despite the fact that the hitter is a substrate of
the enzyme once formed, This result. is in clear
‘contradiction to the first, prediction listed.
‘Monod et at. (051) were the first to subject
these questions to a systematic analysis cmploy-
ing the Pyalictosidase svstem of i. craft, They
reported violations. of the first. two predictions
‘Thus, - pheny4-@-thiogulactoside

-noted above.

[von 63

was found to he a potent competitive inhibitor,
both tn vire and 72 vitro of the B-galactosidase of
B. coli, Despite this, it exhibited no inductive
capacity and in addition aetuaily showed itself
to be an effective inhibitor of enzyme synthesis
in the presence of such inducers as melibiose and
methyl-6-galuctoside.

Interesting information stems from_ the
remarkable results obtained by Pollock (1930,
1952) in his study of penicillinase production by
Bacillus cereus, Were the combination between
the inducer and its cellular co-complexant is
virtually iereversible, As a consequence of this
relative irreversibility, even fleeting exposures of

cells to penicillin at 0 C can fix a sufficient

number of inducer*molecules as to enable the
cells to form euzyme subsequently when. placed
in an environment lacking free inducer. An.
estimation by Pollock, (1952) of the dissociation
constant of ‘penicillin as an inducer yields a
valuc of 3 X 10-7. Pollock (personal communica-
fio) has in unpublished experiments evaluated
the dissociation constant between penicillin and
extracellular penteiilinase and found a value of
GX 10-8, This 200-fold diserepaney between
the dissoviation constants of penicillin as an
inducer and as a substrate of the enzyme would
represent a direct contradiction of prediction (3),
had they both been obtained from intact cell
measurements. The possibility of a change in K,
ralue on isolation of an enzyme makes conclu-
sions drawn from such comparisons to that
extent uncertain. Such modifications on isolation
have in fact been realized in the ease of 3-zalacto-
sidase (Lederberg, 1950) and = malic oxidase
(Gluennekens, 1951). ,

It is the purpose of the present paper to
present experiments relevant to the sceond and
third predietions listed. The system employed
is the induction of maltase in yeast by @-inethyl
glucoside (AMG) under conditions in which -the
latter is unutilizable asia substrate, The data
obtained indicate that the dissociation constant
of -a-methyl. glicoside ns an ie tivo enzyme
complexant is markedly diferent from the value
obtained awhen it is emploved as an inducer,
The data further demonstrate that the low
enpacity of e-methyt glucoside to inhibit maltose
utilization by the induced svstem is nencoinpeti-
tive in nature,
1954]
MATERIALS AND METHODS
Yeast strain, The yeast strain used in. the
_present. investigation is strain K, a diploid

representative of Saccharomyces,
Conditions of culture, Cells were grown ina
complete medium = prepared by adding the
following to one liter of water: bactopeptone,
5g; yeast extract, 2.5 4; sodium lactate (60

%) 6.0 vil; caleinm= chloride, 0.25 ¢; MgSO.”

0.25 IKEAPOs, 2 ¢; (NHNOy, 6 #3 and glu.
cose, JO ve. oy

Cultures were incubated in the above medium
in standing cotton plugged 125 ml Erlenmeyer
flasks, containing 50 .ml of medium. Unless
otherwise specified all inenbations. were carried
out at 30 C, and exponential phase cells were
employed.

Conditions: of induction, In order to obtain -

rapid and effective enzyme synthesis, employing
a-methy! elucoside as an inducer, it was found
desirable to have the eclls suspended in a syn-
thetic medium during the induction, Burkholder's
(1934) meditan was modified hy removing the
asparagin and adding 5.9 ¢ of ruccinie acid to
inerease the buffering capacity. Then the medium
was brought to pH 4.5 by the addition of OTL,
Henceforth this medium will be referred tocas
medium 2. .
Inductions were conducted in medium B with
the addition of various concentrations of glucose
and a-methy! glucoside. They were carricd-out in
large test tubes placed in a water bath at 30 C
with stirring, accomplished) by bubbling cither
air or nitrogen, depending upon whether aerobic
or anaerobic conditions were desired. Ino all
cases the gas was saturated previously with
water vapor, . ‘
Stabilization of enzyme content. To. obviate the
diffienlty of enzyme synthesis in the course of

ass

  

discovery by Swenson and Giese (1950) that
ultraviolet Irradiation stops enzyme formation.
Four ml aliquots were irradinted in open. petri
dishes (7.1 em. in diameter) 15 em from a low
pressure, 15 watt General Eleetric germicidal
bilb, Untess otherwise stated the radiations were
carried out, for 160 seconds, Suspensions were
stirred during trradiation by means of a magnetic
stirrer, Follawing exposure the samples were
ransferred quantitatively with cold water to a
centrifuge tube, harvested, rewashed with cold
water, and suspended in“ ml of cold mediuin B.

THE ROLE OF THE INDUCER

W for enzyme content, use was made of the:

207

The samples were stored then at 3 C fer sub-
sequent assay of enzyme activity. In preliminary
experiments ne detectable loss of enzyme activity
oecurred during storage. The dose of ultraviolet
given to prevent further enzyme formation
reduced the viable. count to Jess) than 0.003
per cent and was of a level such that no photo-
reactivation of the enzyme synthesizing system
was observable on exposure to visible light,

Preparation of suspousion. Cells were harvested
hy centrifugation from the growth medium
innnediately prior to the experiment, They were
washed twice in cold water and resuspended in
medium B to the desired density with the
id of a Klett-Summerson colorimeter previously
calibrated for this purpose. The density employed
was such as to contain 2.6 mg dry weight of
yeast per ml of suspension. .

Manometrie measurements, nayme activity
was estimated manometriedly employing 3) per
cent maltose as substrate, All such measurements
were carried out at 30 C with a standard Warburg
apparatis under anacrobic The
cnayme aclivities are expressed in terms of Qdo.
For reasons which are not as yet clear, complete
expression of maltase content in @-methyl
mlucoside induced, ultraviolet stabilized cells is
not achieved unless small amounts of glucose
are added. 16 was customary to include one mg of
ghicose with the substrate. The level of maltase
activity was estimated over a 50 to 60 minute
period after the glueese added was accounted for
as COn. The QSo. values obtained from intact
cells are in’ excellent agreement with direct
mensurements of maltase activity in dried cell
preparations (Halvorson and Spiegelman, 1952).

Growth during the induction was checked by
both direet and viable counts. Except for one
aerobie experiment. of over 3 hour duration,
no detectable increase in cell number occurred
during the period of the experiment. The reason
for this stems from two facts, One is that ex.
ponential phase cells harvested from the complete
medium and inoculated into thesynthetic medium
B have a Jag of 2.5 hours, The other is that the
densities routinely employed ino the induction
correspond to 2.5 X 10% cells per ml which is close
to the stationary phase level of the organism,

conditions,

RESULTS

Combining constant of cemethyl glucoside as an
inducer af maltase. A study of the kincties of
268

induction with a-methyl glucoside -has been
made and js deserihed elsewhere (Spiegelman
and Halvorson, 1954, unpublished data). The re-
sulls obtained reveal that if the energy supply is
not restricted, the synthesis of maltase is exponen-
tial with time: This makes the determination of
the N, valne of the inducer somewhat more com-
plicated than that encountered in the usual en-
~ayine substrate experiments, It is of some interest,
therefore, to detail the reasoning employed in
making the calculations,
“'The usual derivation of the Michaelis-Menten

-relution «starts with the following reaction
sequence:
hy he :
E+S aa ES—— WwW
t

Here £, S, and P designate enzyme, substrate,
and produet respectively, and the k's represent
the corresponding © réaction — velocities: ‘Two
assuinptions are generally made. One is that the
velocity of the reaction’ is proportional to [4S],
the concentration of the enzyme. substrate
complex, The other is that A: is considerably
smaller than A’. The relation derived from the
equilibrium condition enables one to determine
the dissociation constant, K,, of the ES complex.

To calculate the A, value of inducer as a
stimulator of. enzyme synthesis, we may start
with « similar reaction sequence:

t4+r— at (2)

Where 7 is inducer and # the cellular component

with, which it combines. We need not specify
the nature of m now except to note that combina-
tion between it and inducer is a necessary
prelude to significant enzyme synthesis | in
inducible systems, ,

We assume, as in the case of the Michaelis-
Menten derivation, that the reaction constant
Ay, governing the irreversible destruction. of the
complex, is small as compared with the back
reaction, It is assumed further that the rate of
enzyme formation is proportional to the con-
centration of this complex, [ai], so that

diy

We = A [ri] (3)

Where A’ is constant. The work of Pollock
(1952) and of Pollock and) Torriani (1953)
provides support for bath of these assumptions,

From) the equilibrium condition of reaction

S. SPIEGELMAN AND HARLYN ©, HALVORSON

{[von. 6S

(2), one can solve for the concentration of the
complex, Substituting in equation (3), we have
de vt (4)
dt [i)4+-K, /
where V is the rate of enzyme synthesis at
inducer saturation for fixed [a] and is equal to
A'[r}. AK, is the dissociation constant of the wi
complex, Relation (4) is formally identical to the
Michaelis-Menten equation. [t cannot, however, .
be employed as such since in-our experimental

Al

: tk eo. . . . ” .
system ‘a is not constant with time. Since the
t

inducer employed is not metabolized, [2] is con-

stant in any given experiment, Consequently, ¥
and, therefore, [7] must be functions of time. The
nature of the function to he specified is suggested
by the observation that the kinetics of enzyme
synthesis in yeast under the experimental
conditions employed here possess an exponential
phase, We assume, therefore, that. aw varies ex-
ponentially with time'and inereases in the form
of the zi complex. The Jatter takes account. of
the fact that increase in enzyme forming capacity
does not take place unless inducer is present.
We find: then that ‘

xs myelV F/R (3)

Where V! is the “growth constant” of 3 at
saturating inducer concentrations, a9 the value of
aw at-time zero, and A, is the dissociation constant
of the wi-complex. ,

Substituting (5) in (4), integrating and
evaluating the resulting constant yields: in

E= Re Gta —1) (6)
From equation (7) it is evident that as soon as
oy

fi L ; K, { becomes significantly greater than
unity, Jf is approximated by the product of K
and the exponential term. Under these conditions
we may write

In = ink += - (i)
i

 

Equation (7) can in principle be used to estimate
A, from the slopes at various inducer coneentra-
tions of the linear portions obtained hy plotting
In Jf against time. The use of equation (7), is
of course, restricted to the linear parts of the
semilogarithmic plots, The relation between the
slopes thus determined and the concentrations
1954]

of the inducer is, from equation (7), formally
identical to the Michaclis-Menten relationship.
The usual methods may be employed, therefore,
on the resulting data to determine the A, value of
a-methyl glucoside. as an inducer. Should 3

turn out to be the enzyme itself, the A. so-

- determined should coincide with that obtained in
experiments measuring combination . between
a-methyl glucoside and the enzyme.
We may summarize here, briefly, the details
‘of how such experiments were carried out,
Exponential phase cells were harvested and

washed immediately prior to their use. Then

they were suspended in medium B to a density of
2.84 mg dry weight of cells per ml. Inductions
were conducted at a series of a-methyl glucoside
concentrations in acrated tubes placed in a water
bath held at 30 C. An cnergy source was provided
“in the form of glucose at a concentration of
3.3 mg per ml. It had heen shown in previous
experiments (Spicgelman and Halvorson, 1954)
that this level does. not interfere with the onset or
subsequent progress of maltase synthesis.
Following the addition of the inducer, samples
were removed at intervals, and their enzyme
content stabilized with ultraviolet light as
“described under Methods. The cells then were
washed with chilled water, resuspended in 0.1 Mt
phosphate-suceinate buffer at pI 4.5, and their
enzyme activity assayed manometrically over a
period of GO minutes. At cach a-methyl ghicoside
concentration tested a mininium of three samples
was taken in the exponential period of enzyme
formation for the determination of rate of enzyme
synthesis, It was found that at the low levels of

TABLE 1
Rate constant of aerobic maltase synthesis at carying
concentrations of c-methyl glucoside

 

 

(S) . (v)
CONCENTRATION [hs] RATH CONSTANT

 

OF @-METINL OF MALTASE Sv)
GLUCOSIDE SYNTHESIS.
O14 0.0190 8.10
0.052 0.014 2.80
0.010 0.0168 0.68
0.002 0.0147 ! O.14
0.001 O.0I35 | 0.08

 

Cells were induced aerobically in the presence
of glucose (3.4 my/ml) as energy souree, Rate
constants determined. from the slopes of the
near portions of the semi-log plots of enzyme
activity versus Ume.

“THE ROLE OF THE INDUCER

269.

     

L i i
0 0.05 0.1 015
S
Figure t. A Vineweaver-Burk plot of (8)/(v)
agninst (S) where (S) is the molar concentration of
e-methyl glucoside and v is the rate constant of
maltause synthesis. The data employed are those
of an experiment detailed in table 1,
glucose employed the cells possessed some
constitutive capacity for enzyme formation,
Consequently, rates. on induetor-free controls
were always determined so that the values
obtained in the presence of inducer could be _
corrected. to. those characteristic of. external

‘induction,

Table | summarizes a typieal set of results
obtained in an aerobic induction, and figure 1
exhibits a. Lineweaver-Burk (1934) ‘plot of
the data, From the latter it is evident that
the response of the rate constant of enzyme
formation to inducer concentration is described
hy the Michaclis-Menten relation. The K,
value may be determined -by the usual statistical
procedures involving the method of least squares
(Wilson ef al, 1912). K, -& 20 was found to
be 0.0016 +: 0.0002.

Analogous experiments were carried out to
determine the A, value for inductions carried
out under anacrobie conditions, These experi-
ments differed from the aerobi¢ oncs only in the
fact that nitrogen rather than air was bubbled
through the tubes. The results of a representative
experiment are detailed in table 2. Tt will be
noted from a comparison of tables 1 and 2 that
anaerobiosis in’ the presence of an external
energy supply does not :ffect materially the
rate of enzyme formation, The rates attained
at the various «methyl glueaside concentrations

under the two conditions are comparable. The

data of table 2 yield AY, - 2¢@ of 0.0020 =: 0.0602
which is in good agreement with that found in the
aerobic experiments.

Ky of ecimethyl glucoside as an inhibitor of
maltase. To make the needed comparison it: wes
now necessary to determine the combining
270

TABLE 2
Rate conslanis of anaerobic mualtase’ sunthesis al
recying concentrations of a-wmethyl giveaside

 

 

@ i tw) |
CONCENTRATION GY) 1 KATE CONSTANT ty
OF a METHYVE ' OF MALTASE ' ~
GLNCOSIDE | " |
ra
0.154 _ 6.9229 6.7
0.052 i 0.0173 3.0
0.010 i 0.0157 ' 0.64
0.002 | o.02 | 9 6.15
0.001 »  O.0119 \ 6.05

Cells were indneed under Nain the presence of
glucose (3.3) mg/ml) as an energy source, Rate
constants were deternined:- from the sivpes of the
“Hinear portions of the semi-log plots ¢7 enzyme
netivity versus Gime,

 

capacity of a-methyl] glucoside with the maltose
utilization by various conecutrations of @-methyl
glucoside at differing levels of maltose, A mseaning-
ful interpretation of such data recsires that
the following conditions he ‘satisfied: 1) The

maltase activity of the cells being studied must”

 
 

he rate limiting relative to glucose mtiization,
2) Enayme’ must neither be fortnes ner Jost
during the test. (3) The e-meths) giucoside
must not interfere significantly with the ghuco-
’ymase system.

The first two conditions were met easily by
employing cells which were partials induced
and ostrbilized with ultraviolet. In. cclls so
prepared, the maltose fermenting capacity
remains at a constant level of one-half or Iess the
rate of glucose utilization.

That the third condition is sath
shown by examining the effect of a-methyl
viucoside on the fermentation of limitins ariounts
of glucose. In. order to make the conditions
strictly comparable to the maltose utihzing
inhibition experiments, half-induced, :traviolet
stabilized cells were used. The resting cells
“were washed, resuspended in pll 4.5 Ser, and
the rate at which they anacrubics iis feceented
one mg of glucase in the presence nod 2) -enee of
a-methy] glucoside was followed mone: -ctrically.
Representative results are wiven i Cgure 2.
The jnifial concentration of ghiensc 71.5
per m)) is below saturation, henee, the feocaenta-

‘ton rate begins to drop inumedintecs of42 con-

sor

do was

 

 

 

 

 

ee ermed,
ron ant of
. enlen-

_tinues doing so as the ghieoxe is
For the purpose of plotting, the
glucose remaining at any given tite:

S.. SPIFGELMAN AND TTARLYN O. HALVORSON

 

me

[von 68

lated from the COs evolved during the  cor-
responding périod. There is clearly no evidence
that the presence of a-methy! glucoside has

“any detectable inhibitory cifeets | on glacose
fermentation even at concentrations of jlucose

that are far below the saturation levels,

A deterinination of the A. value of maltose
asa substrate was made and found to be in the
neighborhood of 0.01 as. This relatively low
combining capacity inade it possible to survey

the effects-of a-methyl ghicoside at lower maltose

concentrations than night otherwise have been
possible. In partientar, it was feasible to examine
the ‘region hy which maltose was not saturating
ils enzyme,

As noted cartier, all) the experiments on
inhibition of maltose fermentation with a-methy!
glucoside were earricd out with half-indueed,
ultraviolet stabilized cells, and the rates. were
determined. manometrically at 30° C under
anaerobic conditions,

It ‘beeame apparent .carly that @-methyl

-flucoside was acting as a noncompetitive in-

hibitor since no concentration of maltase could
achieve a complete reversal of the inhibitory
effect. This property of the inhibition is exempli-
fied in figure 38 whieh iHustrates the inverse plot
cammonly employed to distinguish between the
virious tvpes of inhibition (Uimbreit ef al, 1949),
It is evident that at both concentrations depicted
both the slopes and the intercepts are modified

300

 

 

vu dz oe o6 7 9A 19

me O/2 mt

Figure 2. A test of the ability of a-methy!
glucoside (0.155 M) te inhibit glucose fermentation
at limiting concentrations, of the Iatter, Ufalf
shade cireles correspond to fermentation in the
presence of a-methyl glucoside and open cireles
to the controls,
   

7954]
200F ‘O.774 MO
(4-CH4-GLUCOSIDE)
1eGh
1G.0F
14.0}
120}
10.0}
sot O.31 M(%-CH,-GLUCOSIDE)
60+ /
‘4 f
oe On CONTROL
20"

 

 

Lot ft tk ek
Qo 40.80 i20 GO 200
vs

Figure 3. A. douhle inverse plot of rate of
maltose utilization against maltose concentration
in the presence and absence of w-methy! ghicoside
and Hlustrating noncompetitive nature of the
iabibition,

 

 

 

1
— eo
af e ®
~~ °
m05-* ° :
e eo
7s .
oe o-: e
G :
0 19 20 3.0
. ™M |

Figure 4. A Hunter-Downs (1915) plot of the
data of table 8, demonstrating the noncompetitive
ature of the inhibition of maltose fermentation
dyeemethyl glucoside.

aseompared with the controls. This is the type of
rstlt characteristic of noncompetitive inhibition.

Another method (Hunter ahd) Downs, 1945) of

examining this question is te. plot Wy crn
—~ 4%

egaist substeate concentration, where {7} is the
concentration of the inhibitor being tested and @

te 4} + ae : : . ry
8 the fractional activity observed (ic. | where
e

sis the velocity in the presence of inhibitor and
t the rite observed in its absence). In the ease

THE ROLE OF THE INDUCER | . 271

eps wags ae. ,,
_of a competitive inhibitor, [/] roy 8 linear

function of the substrate copeentration, For a
noncompetitive inhibitor,-on the other hatd,

a sos
(1) low independent of substrate concentra-

tion. That this is the situation in the present
case can be seen from figure 4 which summarizes
data obtained at seven diferent concentrations
of maltase and three concentrations of a-methyl
glucoside,

As shown by Hunter and Downs (1945) the
dissocintion constant Ay. of a noncompetitive
inhibitor possessing the properties indicated in

 

TABLIE*
The determinalion of the dissociation constant (Ky)
of cemethyl glucoside and the maltose
utilizing enzyme

    

 

 

 

 

 

 

EXPT a-aterat | MALTOSE a

cs iar ed | i

1 | 0.0775 0.007 | a. | 0.70

O.0N | OSB: . 0.84

0.10 0.932 | 0.34

0.20) OSS | 0.56

0.50 1 O77 | 0.26

1.00 | 0.85 | 0.38

'

2 10.0775; 3.00 1 O.8s8 3 0.61

oo boss | ose

0.20 0.76 | 0.25

0.75 | 0.7535 | 0.32

1.50 1) 0.786 4 0.29

3.0) | 0.815 | 0.54

3 10.195 | 0.0 | ost 0.77

. 0.20 | 0.680 5 0.86

0.758 | 0.747 | 0.48

1.80 1 O80 4 0.60

1 3.000 | 0.727 ; 0.43

4 j 0.310 | o.c0 | ons ! 0.70

| 0.75 | O55 | 0.88

PESO E OSet | 0.42

: 8.00) 2 UG1R § Olds
Menn..... doves eebeedeeecvenes 0.418 0.057"

* Two standard deviations of the mean, Youn.

Anaerohic nialtose fermentation was followed
minometriesliy at 30°C. The eells were half-
imfluecd and wltravielet stabilized. Vy is the rate
is the rate in its
ahsence. Ay was ealculated using equation ($8).

in the presence of inhibitor and 3
272 S. SPIRGEEMAN
figure 4 can be determined from the following
relation:

Ke = Ul} i “- (9)

where the symbols have the meanings mentioned
above: The results of 21 such determinations
are summnarized.in table 3, from whieh it is seen
that an average value of 0.46 was found for K;.

DISCUSSION

The experiments described in the present
paper were designed to see whether inducer
coinbined specifically with existing enzyme in
inducing the synthesis of new enzynie molecules,
This was accomplished in terms of a comparison
of the dissociation constants of «- -methyl gluco-
_side as an inducer of maltase synthesis and asa
complexant with the enzyme. An average A,
value of 0.0020 mM was found for its induetive
activity anaerobically. This is to be compared
with O46 Mas its dissociation constant with
enayine. Evidently a-methyl pluecoside has a
250-fold greater affinity for whatever structure it
combines within its role as an inducer than it
has for the enzyme molecule, the synthesis of
which it ean induce. These results are not in
agreement with any simple interpretation of the
often suggested mechanism of induction which
requires that the inducer function as. 2 specific
complexant with -enzyme as a. primary and
mandatory step in the induced synthesis, of
enzyme, ,

Experiments with two: systems have, by now,
provided interpretable data relevant to the role
of the inducer in enzyme synthesis. They are
B-galactosidase synthesis in #. coli (Lederberg,

1951; Monod ct al., 1951) and maltase formation
in 8S. cerevisiae. These - investigations have
sueceeded in violating one or more of the pre-
dictions derivable from the complexing concept.
Under the. circumstances it seems advisable to
abandon this interpretation of the role of inducer,

SUMMARY

a-Methyl glucoside, an analogue of maltase,
can induce the synthesis of nialtase in Sae-
charomyees cerevisiae. Fxperiments were per-
farmed designed to see whether a-methiyl
slreoside combined with maltase in the course of
stimulating its. synthesis, The dissociation
constant of a-methyl glucoside as an inducer

AND HARLYN 0. HALVORSON

{von. 68

was nieasured and found to be 0.0016 s1 4: 0.0002
aerobically and 0.0020 at +: 0.0002 anaerobically,
Measurement of the dissociation constant
between. e-methyl ghicoside and the maltase
utilizing enzyme pave a value of 0.46 a. The
marked diserepancy between — these values
indicates that a-methy} glucoside does not.
combine with-maltase in the process of stimulat-
ing the syrithesis of this enzyme, OS

These results, along with others on the synthe.
sis of B-yalactosidase, suggest the necessity of
ahandoning the complexing coneept to explain
the role and specificity of inducers in enzyme
synthesis,

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