AN ABSTRACT OF THE THESIS OF
Kemet Dean Spence
for the
(Name)
Date thesis is presented
in Microbial Physiology
(Major)
(Degree)
Ph. D.
May 14. 1965
Title REGULATION OF THE TERMINAL REACTIONS
FOR METHIONINE BIOSYNTHESIS IN YEAST
Abstract approved
Redacted for Privacy
,- .. ajor professor)
Studies were carried out to elucidate the mechanism of enzy-
matic control of methionine biosynthesis in Saccharomyces cerevisiae.
Enzymatic studies demonstrated that, in addition to the methionine activating enzyme, the S- adenosylmethionine:homocysteine
transmethylase enzyme was induced during cultivation in the presence of excess methionine. A similar, though reduced effect was
observed when homocysteine and serine were present in the medium.
The serine hydroxymethylase enzyme was
repressed under these
conditions.
Ethionine- resistant mutants were isolated to facilitate these
studies. These organisms were shown to possess a reduced ability
for converting methionine to S- adenosylmethionine. These organ-
isms accumulated more intracellular methionine than the wild -type
strain.
The mutants were also found to excrete more methionine into
the medium than the parental strain.
All mutants tested showed a
reduced level of the transmethylase enzyme.
Two showed a
re-
duced level of the methionine activating enzyme.
Studies which measured the accumulation and intracellular dis-
tribution of ethionine (ethyl-C14) and methionine (methyl -C14) were
also carried out. The mutants were able to exclude' the analog from
participation in most cellular processes.
REGULATION OF THE TERMINAL REACTIONS
FOR METHIONINE BIOSYNTHESIS IN YEAST
by
KEMET DEAN SPENCE
A THESIS
submitted to
OREGON STATE UNIVERSITY
in
partial fulfillment
of
the requirement for the
degree of
DOCTOR OF PHILOSOPHY
June 1965
APPROVED:
Redacted for Privacy
Associate Professor of Microbiology
In Charge of Major
Redacted for Privacy
Chairman of Department of Microbiology
Redacted
for Privacy
Dean of Graduate School
Date thesis is presented
Typed by Muriel Davis
May 14, 1965
ACKNOWLEDGMENT
Parks:
To Dr. Leo W.
.
.
.
.
.
.
.
.
.
.
.
.
for his aid in interpretation and scientific
development.
for his keen sense of responsibility
for his students.
for a million other things, too numerous
to mention.
To the faculty, staff and fellow graduate students:
.
.
.
,
for their assistance.
To the world of
.
.
.
literature and the American Indian:
.
for filling the third and fourth quadrants.
To my wife:
.
.
.
.
for love.
.
.
.
for staving off starvation.
TABLE OF CONTENTS
Page
INTRODUCTION
1
REVIEW OF LITERATURE
4
MATERIALS AND METHODS
Cultures
Cultures and Media
Microbiological Assay for Methionine
Protein Determinations
Isotope Measurement
Ion -exchange Columns
Cell -free Extracts
S -adenosylmethionine:homocysteine Transmethylase
Assay
AM- synthetase Assay (Methionine Activating Enzyme)
Serine Hydroxymethylase Assay
Supplements and Enzyme Assays
Selection of Mutants
Intracellular Distribution of Methionine and Ethionine
Growth in Methionine and Ethionine
11
11
11
12
12
12
13
13
13
14
14
14
15
16
17
RESULTS
18
DISCUSSION
50
SUMMARY
56
BIBLIOGRAPHY
57
LIST OF FIGURES
Figure
1
2
3
Page
Growth during exponential phase with strain
3701B in supplemented and non - supplemented
WCLM media
20
Growth during exponential phase with strain
J2F1 in supplemented and non - supplemented
WCLM media
21
Growth during exponential phase with strain
R3720 in supplemented and non - supplemented
WCLM media
4
5
6
7
8
9
22
Growth during exponential phase with strain
A8E90 in supplemented and non - supplemented
WCLM media
24
Endogenous level of methionine during growth
in the absence and presence of homocysteine
plus serine (Hc S)
25
Accumulation of methionine in the medium during
growth in the absence and presence of homo cysteine and serine (Hc. S)
27
The accumulation of methionine during incubation
in the presence of 1mM 1- methionine
29
In tracellular methionine concentration during
incubation in 1 mM 1- methionine
30
Disappearance of endogenous methionine during
incubation in the presence of mM 1- methionine
31
Proposed methionine biosynthetic pathway in
Saccharomyces cerevisiae
33
Serine hydroxymethylase activity in strain 3701B
during exponential growth in supplemented and non supplemented WCLM media
34
1
10
11
Page
=r1lr.^-_.
12
13
14
15
16
Transmethylase activity in strain 3701B during
exponential growth in supplemented and non supplemented WCLM media
36
AM- Synthetase activity in strain 3701B during
exponential growth in supplemented and non -
supplemented WCLM media
37
Serine hydroxymethylase activity in wild -type and
mutant strain after cultivation in supplemented
and non - supplemented WCLM media
38
Transmethylase activity in wild -type and mutant
strains after cultivation in supplemented and
non - supplemented WCLM media
40
AM- Synthetase activity in wild -type and mutant
strains after cultivation in supplemented WCLM
media
41
LIST OF TABLES
Page
Table
1
2
3
4
5
6
Concentration of Labelled Methionine and Ethionine
in the Expandable Pool
43
Concentration of Labelled Compounds in the Internal
Pool after Incubation in Isotopic Methionine and
Ethionine
44
Concentration of Label in Hot TCA- soluble Fraction
After Incubation in Methionine -C 14H3 and EthionineCH2C 14H3
46
Incorporation of Labelled Methionine and Ethionine
into Protein
47
Incorporation of Label from Methionine and Ethionine
into Sterols
48
Net Accumulation of Labelled Methionine and Ethionine
after Cultivation in the Amino Acid and Its Analog
49
REGULATION OF THE TERMINAL REACTIONS
FOR METHIONINE BIOSYNTHESIS IN YEAST
INTRODUCTION
The ecological advantage afforded an organism which exhibits
the greater economy of cellular processes is obvious. Organisms
which could not competitively exist and suffer the evolutionary flux
would no longer survive. Many organisms have been found which ex-
hibit the subtle and beneficial ability to rapidly adjust intracellular
processes to environmental changes.
Control mechanisms in living organisms have now become
axiomatic owing to a comparatively recent flurry of research activity
directed towards determining the character of macromolecular regulation and structure. The intracellular aristocracy is of course dominated by DNA. The genetic information contained therein is passed
to RNA, via
transcription, from whence the information is further
translated into protein. Since proteins are the primary acceptors of
absorbed compounds, and catalyze the chemical reactions of the cell,
the levels which they assume and the activity which they exhibit are
of
paramount importance to the survival of the organism.
At the present time, two phenomena affecting enzyme level and
activity have been recognized and extensively investigated. These include the alteration of enzyme function, or feedback inhibition, and
z
the suppression or stimulation of the synthesis of new protein, known
as enzyme repression or induction, respectively. These controls do
not apply to all enzymes.
One is tempted in these instances to con-
clude, perhaps presumptuously, that this apparent lack of control
exists only in the presence of stresses exceeding or extrinsic to those
encountered during their evolution. It would be even more arbitrary
to assume that all facets of enzyme regulation have been satisfac-
torily resolved.
The great bulk of information serving as a basis for current
hypotheses of metabolic regulation has been derived from studies
with bacteria. These comparatively simple organisms are quite dif-
ferent, however, from the higher life forms. The yeast cell, commonly considered a microbe quite similar to the bacterium, in actu-
ality resembles the mammalian cell in many respects. As in many
higher forms, the yeast not only has a stable haploid state, but also
mates with the opposite sexual type to form a stable diploid cell,
which under certain conditions may, via meiosis, return to the haploid
state. At the present time eighteen chromosomes have been postulated in yeast. Unlike the bacterial cell, the genes cooperating within a given biosynthetic or catabolic pathway are
rarely found in juxta-
position, but are most often unaccountably scattered throughout the
chromosomes with perplexing irregularity.
Contrary to the circumstance of bacteria, yeast control
3
mechanisms have not been extensively investigated.
Although there
are certain advantages inherent in the simplicity of bacteria, there
are also advantages in employing a more complex organism. In
yeast, one is aided by their great metabolic versatility, ease of cul-
ture, and their well- defined Mendelian genetic mechanism.
With these tools in hand, studies were undertaken with Saccharo-
myces cerevisiae to attempt to ascertain the effect of certain methionine biosynthetic intermediates and methionine analogs on the control
of the enzymes involved in the
bio synthetic pathway.
terminal reactions of the methionine
4
REVIEW OF LITERATURE
Repression was first discovered in
1953 when Monod and Cohen -
Bazire found that the formation of the enzyme tryptophan synthetase
was selectively inhibited by tryptophan and certain tryptophan analogs
in Escherichia coli.
As defined by Vogel (1957a) enzyme
decrease in the rate
of
"repression" is a relative
synthesis of particular apoenzymes while "in-
duction" is relative increase in the rate of synthesis of particular
apoenzymes. It is in this context that these terms will be used here.
Since the original discovery, many other cases of enzyme re-
pression have been detected and studied. These include repression
of 1- threonine
deaminase by 1- isoleucine (Umbarger and Brown, 1958),
aspartokinase by
Szulma jster and
1- threonine (Stadtman, et al.
Co
r r i v a u x,
1
9 6
3;
1
9 6
,
1961; de Robichon-
4) methylation of 1,
homocysteine by 1- methionine (Rowbury and Woods,1961; Wijesundera
and Woods, 1960), eight enzymes of the arginine biosynthetic se-
quence (Gorini, Gundersen and Burger, 1961; Vogel, 1957b; Bacon
and Vogel, 1963), alkaline phosphatase by orthophosphate (Torriani,
1960), the histidine biosynthetic enzymes by histidine (Ames and
Garry, 1959; Ames and Hartman, 1963), and of enzymes involved in
pyrimidine biosynthesis (Yates and Pardee, 1957).
In 1961, Jacob and Monod formulated their hypothesis for
5
enzyme induction.
Since that time the hypothesis has passed through
continual metamorpheses, and now encompasses both inducible and
repressible enzyme systems. The existence
of specific
regulatory
regions as well as elements conferring specificity for protein struc-
ture in the genomes appears to have been well founded; in this respect
most systems can be made to more or less conform to the general
mechanism of control postulated by those investigators.
In the arginine investigations, all eight arginine biosynthetic
enzymes were simultaneously repressed when the organism, E. coli,
was grown in the presence of this amino acid (Bacon and Vogel, 1963).
This anabolic system, in addition to expressing the characteristic
of
coordinate repression, gave impetus to the unified theory for in-
duction and repression (Vogel, 1957b; Bacon and Vogel, 1963), es-
pecially when it was discovered that mutations of the repressor gene
arose which made the enzyme acetylornithine
ducible.
-transmethylase in-
The other seven enzymes of the system retained their nor-
mal repressible character.
Constitutive mutants for alkaline phosphatase, normally re-
pressible by orthophosphate in the wild type organisms have been
isolated (Torriani, 1960). These mutations have been found to be located some distance from the gene responsible for the enzyme, and
have been shown to be recessive.
Mutations similar to those of the operator gene in the inducible
6
system were reported for Salmonella typhimurium. Since the original
discovery that four enzymes of the histidine biosynthetic sequence
were coordinately repressed by histidine (Ames and Garry, 1959),
the histidine system has received considerable attention. Both feed-
back and repression exist in the system; in addition, what are termed
"polarity" mutants have been isolated (Ames and Hartman, 1963). The
discovery of polarity mutants has led to the term "modulation ", or the
control of the reading of the histidine messenger. The enzymes of
the histidine biosynthetic sequence occur in a cluster on the bacterial
chromosome, and code for ten known structural genes. Polarity mutants arise within this genetic area, and are termed thus since
all
en-
zymes to the left of the mutation are produced at reduced levels (di-
rection arbitrary). Enzymes coded in genes to the right of the point
mutation are produced in normal quantities. It has been postulated
that polarity mutations cause an alteration in the efficiency of translation from messenger RNA into protein.
Microorganisms other than bacteria have not yet received such
extensive investigation; however, it has been sufficient to conclude
that control systems exist in the higher organisms which are similar
to those of
bacteria.
In Neurospora
crassa, although the formation of the terminal
enzyme in the tryptophan biosynthetic sequence, tryptophan synthe-
tase, was not markedly affected by tryptophan, and only slightly
7
by the analog, 6- methyltryptophan (Lester, 1961a), an
earlier en-
zyme (indole synthesizing ability), was reduced to non -detectable
levels under the same conditions (Lester, 1961b). It has also been
reported that there is slight repression of pyrroline -5- carboxylate
reductase by proline (Yura and Vogel, 1959). Repression was not detected by Ames and Garry (1959) in the histidine enzymes of this organism, nor in shikimic acid synthesis (Gross and Fein, 1960).
The occurrence of
repression in yeast was first reported by
Stadtman and coworkers (1961) when it was found that aspartokinase
synthesis was repressed by 20 mM 1- threonine. This enzyme and
two others involved in the
early reactions of the biosynthetic system
shared by threonine and methionine, homoserine dehydrogenase and
semi -aldehyde aspartic dehydrogenase, were later investigated by de
Robichon -Szulmajster and Corrivaux (1963; 1964). It was found that
threonine repressed aspartokinase and semi-aldehyde aspartic dehy-
drogenase by
10
75 and 10
percent, respectively, at a concentration of
mM, but did not alter the level of homoserine dehydrogenase.
Methionine on the other hand, repressed aspartokinase and homo-
serine dehydrogenase, but amazingly, stimulates an increase of
51
percent in the level of the third enzyme. This latter phenomenon
is similar to that reported by Spence (1962) for the methionine-
synthesizing ability in yeast.
Another interesting occurrence in yeast was reported by Pigg,
8
Sorsoli and Parks (1964) where it was found that the enzyme leading
to the formation of S- adenosylmethionine via methionine was inducible.
In fact, the organism does not appear able to
curtail the production
of
the endproduct even after huge quantities have been accumulated.
Both endproduct and enzyme synthesis continue at a high rate until
methionine is exhausted from the medium. In another yeast, Candida
utilis, Svihla and Schlenk
(
1960) have shown
that the large S- adenosyl-
methionine pool which accumulates in the vacuole is not available for
further cellular processes, even under conditions of sulfur starvation, and only disappears via dilution through cell division.
The phenomenon
referred to as "negative feedback" has been
found to occur in many microbial systems (Umbarger, 1961).
In E.
coli three apparently different aspartokinases were found to exist
(Stadtman, et al.
,
1961).
One was specifically and non - competitively
inhibited by lysine; a second was specifically and competitively inhibited by threonine.
The existence of a third aspartokinase, in-
hibited by homoserine was also found. The condensation of phospho-
ribosyl- pyrophosphate with adenosinetriphosphate has been shown to
be strongly inhibited by histidine, the endproduct in this pathway in
Salmonella typhimurium (Ames, Martin and Garry, 1961; Martin,
1963).
In Neurospora, it has been shown that preformed indolesyn-
the sizing activity was inhibited by 1- tryptophan, 4- and
9
6- methyltryptophan.
The inhibition was at least partially competitive
since the affects were somewhat reversed by the presence of the indole precursor, anthranilic acid (Lester, 1961b).
In Saccharomyces, the aspartokinase enzyme, shared by two
biosynthetic endproducts, methionine and threonine, and their key
intermediate, homoserine, were not only repressed by these compounds, but also inhibited(de Robichon -Szulmajster and Corrivaux,
1963).
Inhibition of the enzyme by methionine was however, subject
to the condition that the organism had been cultivated in a medium
containing methionine.
The use of amino acid analogs has been of great value in the
study of control mechanisms. Many mutants resistant to the effects
of normally inhibiting concentrations of
certain analogs have been
found to be non -repressible by the corresponding amino acid.
Mu-
tants of E. coli have been isolated and studied which were insensitive
to the presence of 5- methyltryptophan and were found to be non -re-
pressible by the endproduct, tryptophan (Cohen and Jacob, 1959).
Other instances of amino acid analogs exerting repressive effects
have been widely reported. It has been found that in addition to 1-
tryptophan, 6- methyltryptophan repressed the formation of tryptophan synthetase in E. coli (Lester and Yanofsky, 1961). Repression
by methionine analogs has been shown to occur in the same organism
(Rowbury and Woods, 1961).
Although varying in their repressing
10
ability, those shown to be effective repressors of methionine syn-
thesis included methionine sulfoxide, methionine sulfone, ethionine,
S- methylcysteine, S- methylmethionine, norvaline and norleucine.
11
MATERIALS AND METHODS
Cultures
Wild type and mutant strains of Saccharomyces cerevisiae were
used in this study. Cultures of strain #3701B were obtained from Dr.
H. L.
Roman, Department of Genetics, University of Washington.
Strain #3701B,
a haploid auxotroph
requiring uracil, served as the
parent strain for all analog insensitive mutants isolated for this work.
Analog
resistant mutants were selected for their ability
to overcome
ethionine inhibition in the presence and absence of homocysteine and
serine.
Stock cultures of all yeast cultures were maintained on yeast
complete (YC) medium (Starr, 1961).
Cultures and Media
When available, all chemicals were obtained from commercial
sources and used without further purification. S- methyl- l- methionine was prepared using the technique of Toennies and Kolb (1945).
L- homocysteine employed in the transmethylase assay was prepared
prior to each experiment by adjusting the desired amount of 1 -homocysteine thiolactone to pH
then readjusted to pH
7. 0
8. 5
with
1
N KOH
with IN HC1.
for ten minutes. It was
12
Wickerhams synthetic medium less methionine (Wickerham,
1946) was
employed as the basic medium for all experiments. This
is referred to in the text as WCLM medium.
Microbiological Assay for Methionine
Methionine was determined using the methionine requiring
strain
of
Streptococcus faecalis, #9790. Growth response was meas-
ured on the Colman Model
9
Nephelo- Colorimeter.
The final
results
were expressed as net millimicromoles methionine formed per milli-
gram dry weight of cells.
Protein Determinations
Protein concentrations of the cell free extracts were determined
according to the method of Lowry, et al. (1951). Crystalline bovine
albumin was employed as the protein standard.
Isotope Measurement
Assay of radioactive isotopes was carried out in a Model 3000
Packard Tri -Carb Liquid Scintillation Spectrometer. The scintillator employed consisted of 0.4 percent
0. 02
percent
1,
2, 5- diphenyl
oxazole (PPO),
4- bis- 2- (5- phenyloxazolyl)- benzene (POPOP) in 48
percent absolute ethanol plus
52
percent toluene.
13
Ion -exchange Columns
Short ion -exchange columns (0.
5
x 2.
0
cm) of either the lithium
or ammonium form of Dowex 50W -8X were employed.
Cell -free Extracts
Enzymatic extracts were prepared by rupture of the frozen cell
mass in an Eaton press under a pressure of
10, 000
lbs. per sq. in.
The crude preparations were then thawed and centrifuged to remove
cell debris. They were passed through four layers of cheese cloth
to remove the lipid layer, and dialyzed in 0. 02M buffer for 20 hours
at 2oC. The pH and type of buffer corresponded to that employed in
the assay.
S- adenosylmethionine:homocysteine Transmethylase Assay
Assay of the transmethylase enzyme was performed according
to the technique of Shapiro and Yphantis (1964) with modification in
the reaction mixture and the ion -exchange columns. Instead of the
lithium form, the ammonium form of Dowex 50W -8X resin was employed. More than twice the recovery of methionine could be obtained
with the ammonium column.
altered to contain
The reaction mixture was only slightly
20 p.moles 1- homocysteine, 20
1- methionine (methyl -C14), and 0.
1
µmoles of S- methyl-
p.mole of Zn + +, in 0. 1M
14
phosphate buffer at pH
7. 0 in a
total volume of
1. 0
ml.
AM- synthetase Assay (Methionine Activating Enzyme)
AM- synthetase was assayed in dialyzed cell -free extracts with
1- methionine
(methyl-C14), according to the procedure of Pigg,
Sorsoli and Parks (1964) with slight modification. In addition to the
lithium columns described, both the lithium and ammonium forms of
Dowex 50W -8X
resin were employed on the short
(0.
5
x 2.
0
cm)
columns.
Serine Hydroxymethylase Assay
Serine hydroxymethylase activity was observed using the method
of Whiteley (1960), with the
modifications of Pigg (1962). Formalde-
hyde was determined by the method of Nash (1953) using monomethylol
dimethyl hydantoin as the formaldehyde standard.
The standard was
obtained from Dr. H. R. Whiteley.
Supplements and Enzyme Assays
The enzyme levels were measured after cultivation in the
pres-
ence and absence of 1- homocysteine plus 1- serine, and in 1- methio-
nine.
In the experiments comparing the enzyme level of the mutants
to that of the wild -type, the supplements were added to actively
15
growing,
18
hour cultures. Incubation was then carried out for an
additional two hours.
The cells were then harvested, washed, and
the cell -free extracts prepared.
In experiments examining the parental
strain alone, supplements
were introduced during exponential growth, two hours prior to har-
vest. When "continuous methionine" treatment was desired, the
amino acid was also added four, six, and eight hours prior to harvest.
Supplements were always added in amounts which would result
in a final concentration of
1
mM.
Selection of Mutants
The parental strain, #3701B, was initially treated with
violet irradiation according to the technique of Spence
of exposure was 40 seconds.
(
ultra-
1962).
Length
An aliquot was then removed from the
irradiated medium, washed, and transferred to WCLM supplemented
with the analog and 1- homocysteine thiolactone and /or serine. Serial
transfer was carried out in the same medium at the desired intervals.
The number of repetitions varied with the mutant group.
The final tube in each
series was centrifuged, washed in dis-
tilled water, and spread on pre -poured plates of
WC to
3
parts Methionine Assay Medium [ Difco]
WC -MAM (12
).
parts
The plates had
been pre- seeded with the methionine indicator strain of Streptococcus
faecalis,
#9790.
Yeast suspensions were routinely adjusted to give
16
approximately 40 -50 colonies per plate. After spreading, the plates
were incubated at 30°C and observed for halo formation by strain
#9790 around the yeast colonies.
Those colonies which stimulated
strong halo formation were tentatively assumed to be producing large
amounts of extracellular methionine, and were isolated for further
study.
Yeast mutant strain #A8E90 was not isolated by this technique.
It was selected from the surface of an ethionine supplemented WM
agar medium. Its full derivation has been described (Spence, 1962).
Intracellular Distribution of Methionine and Ethionine
Each strain was inoculated into WCLM medium and incubated
for
18
hours at 30oC. They were then centrifuged, washed in distilled
water, and redistributed in fresh WCLM media containing either,
1
mM 1- ethionine -C 14, (2)
teine thiolactone and
or (4)
1
1
1
mM 1-ethionine plus
mM 1- serine, (3)
1
1
(1)
mM 1-homocys -
mM 1- methionine -C
14
H3,
mM 1-methionine-C 14H3 plus 1-mM 1-homocysteine thiolac -
tone and 1- serine. Cell concentrations were adjusted to give approxi-
mately
1. 5
mg dry weight of cells per ml.
At this concentration the
cells are capable of accumulating no more than
20
percent of avail-
able supplement during the experimentally allotted time.
The cells were allowed to incubate in the supplemented medium
for four hours. At the end of this time the cells were centrifuged,
17
and a sample of the supernatant removed for assay of label remaining
Expandable pool, internal pool, and protein concen-
in the medium.
trations of radioactive substances were measured according to the
technique of Kempner and Cowie (1960)
.
Assay of the hot TCA-
soluble fraction (containing the nucleic acids) was accomplished after
separation according to the procedure of Schneider (1945). Total
sterol was recovered from the cells using the method of Starr and
Parks (1962).
Growth in Methionine and Ethionine
Growth curves for the wild -type and mutant clones were meas-
ured in WCLM media supplemented with either;
1- homocysteine
cysteine plus
ethionine,
2
thiolactone plus
1- homocysteine
ethionine, or
mM 1- serine, (3)
1
mM dl- ethionine, (4)
(5) 2 mM
(8)
dl- ethionine,
thiolactone, plus
1
(1) nothing, (2)
(6)
1
1
1
1
2
mM dl-
mM 1- methionine, (7)
mM 1- methionine plus
2
2
mM
1
mM dl-
mM dl- ethionine.
Tur-
bidity measurements were made periodically throughout the first
three hours of the exponential growth phase on the Colman Model
9
Nephelo- Colorimeter.
mM
mM 1 -homo-
mM 1- serine plus
mM 1- serine, and
1
18
RESULTS
Representatives of two different mutant groups were isolated in
these studies. In the first group, designated as the J2F Series, mutants were selected after seven serial transfers through WCLM medium supplemented with 100 µgm per ml dl- ethionine and
O.
1
mM 1-
serine. Mutant strain #J2F1 was chosen to represent this group since
it accumulated the highest level of extracellular and intracellular
methionine.
In a second group, the R37 Series, two
strains, showing exten-
sive halo formation on feeder plates, were selected after three serial
transfers through WCLM medium supplemented with
dl- ethionine plus
2
600 µgm
mM 1- homocysteine thiolactone and
1
per ml
mM 1-
serine. Strain #R3720 was arbitrarily chosen for more extensive
investigation.
In addition to these
strains, strain #Á8E90 was also included in
these experiments. Its derivation has been described earlier.
All mutant strains and the parental strain #3701B were tested
for their ability to grow in the presence and absence of ethionine,
and in media supplemented with those methionine biosynthetic inter-
mediates which could be expected to alter sensitivity to the analog.
These results are in Figures 1 -4. It should be noted that the dry
weight equivalent of cells was calculated to reach a maximum of
19
3
x 10 -2 mg per ml.
At this cell concentration the levels of amino
acid supplements are not significantly altered during the course of
the experiment.
The
characteristic growth response
of the wild -type
#3701B under these conditions is shown in Figure
1.
strain
Maximum
growth is obtained in the non - supplemented medium and nearly the
same value is obtained when homocysteine and serine are added.
Methionine is seen to nullify the effects of ethionine while concomi-
tantly possessing its own suppressive effect. Homocysteine and
serine, alone or in combination, exhibit a slight reversal of ethionine
inhibition.
Strain #J2F1 exhibits similar responses under these conditions.
This organism is shown in Figure
2.
The rate of growth is, however,
somewhat slower in the non - supplemented or homocysteine and serine
supplemented media. The slight advantage obtained during cultivation
in the presence of ethionine, supplemented with homocysteine and /or
serine, is apparently sufficient to allow competitive selection of this
mutant.
With
strain #R3720, shown in Figure
3,
it can be seen that not
only does methionine lack the suppressive effect, but only in the
presence of this amino acid is a growth rate obtained which is comparable to that of the parental strain. Without methionine the rate
is significantly reduced. It can also be seen that homocysteine and
20
N
H
S
E
M
Non- Supplemented
1mM
1mM
2mM
1mM
Homocysteine
Serine
Ethionine
Methionine
0. 140
4-)
a'
0. 120
N
H. S
.9
,a)
0. 100
P
a)
z
o
O.
080
EM
ea)
o
]
.,
rd
M
0. 060
0.040
H E; S E;
U
H
0. 020
H.
E
Unit Time (Hour)
Figure
1.
Growth during exponential phase with strain
3701B in supplemented and non - supplemented
WCLM media.
SE
21
N
H
S
E
M
Non -Supplemented
1mM Homocysteine
1mM Serine
2mM Ethionine
1mM Methionine
0. 140
-1..>
.r-,
Z
0.120
o
a
R,
N; H' S
0. 100
aD
z
°
bp
0.080
o
a
O
M;
0.060
EM
a.)
ca
0
0.040
S- E
U
H.
Ho
H
0. 020
E
SE
E
Unit Time (Hour)
Figure 2. Growth during exponential phase with strain
J2F1 in supplemented and non -supplemented
WCLM media.
22
N
H
S
E
M
Non- Supplemented
1mM
1mM
2mM
1mM
Homocysteine
Serine
Ethionine
Methionine
0. 140
w
"E
0.120
M;
EM
0.100
N;
H
o
J0
.
0.080
H'SE
0.060
SE
HE
bo
o
a0
S
...,
a)
cn
ái
0.040
U
0
'-I
0.020
Unit Time (Hour)
Figure
3.
Growth during exponential phase with
strain R3720 in supplemented and non supplemented WCLM media.
23
serine, alone or in combination, reverse the repressive effect of eththionine.
Homocysteine is most effective in this respect, while addi-
tional advantage is afforded the cell when serine is also present. In
addition, the effect of ethionine alone is reduced considerably below
that of the parental strain.
The effects on the growth of strain #A8E90 are shown in Figure
4.
Growth of this organism in the non - supplemented medium is the
same as for the wild -type. The response in media containing homo-
cysteine and serine, or homocysteine plus serine plus ethionine, is
the same as that for the homocysteine plus serine supplemented medium of the parental strain.
Homocysteine or serine significantly re-
verse the effects of ethionine. The effect of ethionine alone is much
less pronounced in this strain than in the wild -type. Other curves
are similar to that of the parental strain.
The endogenous level of methionine acquired during growth in
non - supplemented and WCLM media supplemented with
cysteine and
1
mM serine is shown in Figure 5.
1
mM homo-
After six hours
the intracellular levels of methionine in strains #A8E90 and #R3720
are approximately twice those found in strain #J2F1 and the parental
strain, irrespective of supplements. Although strain #A8E90 attains
levels above #R3720, it apparently activates the AM- synthetase enzyme which then depletes this pool. Strain #R3720 does not react
in the same manner, but maintains a consistently high level of
24
N Non -Supplemented
H 1mM Homocysteine
1mM Serine
E 2mM Ethionine
M 1mM Methionine
S
0. 140
4J
.r,
0.120
0
N
0
aa)
z
o
0.100
HS
0.080
H E; S E
HSE
EM
tin
o
a
.,
É; M
0.060
o
m
ea
a
0.040
U
H
0.020
Unit Time (Hour)
Figure 4. Growth during exponential phase with
strain A8E90 in supplemented and non supplemented WCLM media.
25
3701B (Hc S)
o--- 3701B
°
o
° A8E90 ( Hc S)
X
X
O
*--4
A
,A
L--d
A8E90
J2 F 1 (Hc S)
J2 F 1
R3720 (Hc S)
R3720
5
2
3
4
Time (Hours)
Figure
5.
Endogenous level of methionine during growth
in the absence and presence of homocysteine
plus serine (Hc S).
26
intracellular methionine. Strain #J2F1 acts in a manner similar to
that of #A8E90, though at lower intracellular methionine concentrations. Its advantage over the parental strain #3701B is of a transi-
tory nature in both media; however, more methionine was available
for cellular processes during the first five hours. The "feeder"
characteristics of the mutants are clearly seen when one observes
the levels of methionine excreted by these organisms into the media.
These results are shown in Figure
6.
After six hours, #R3720 has raised the methionine concentration in the medium to
O. 9
rnp.moles per mg dry weight of cells.
This
is approximately 20 percent higher than that attained by #A8E90 when
homocysteine and serine are both available, and
30
percent higher
than when in the non - supplemented medium. The parental strain,
#3701B showed the lowest level of
extracellular methionine acquired
during incubation in non- supplemented WCLM medium; this value is
about 38 percent below that of #R3720 under the same conditions.
Values for other samples fell between
O.
68 and
O. 7
mµmoles per mg
dry weight cells. When the gradient between intracellular and extra -
cellular methionine is compared, one finds an important relationship.
In non - supplemented medium the ratio between intracellular and
extracellular methionine in the wild -type is
4. 4:1.
cysteine and serine supplemented medium is
2. 8:1.
That in homoWith the mu-
tants, values obtained for both media are nearly identical:
5. 4:1
27
3701B (Hc S)
C
fl 3701B
o A8E90 ( Hc S)
o-----0 A8E90
o
X
X
-X
X
A
A
J2F1 (Hc S)
J2F1
R3720 (Hc S)
R3720
0.9
0.8
0.
7
Methionine
v
o an
a)
O. 6
IlE
0.2
0.
1
/
/
1
1
2
3
4
5
6
Time (Hours)
Figure
6.
Accumulation of methionine in the medium during
growth in the absence and presence of homocysteine
and serine (Hc S).
28
and
5. 1:1
for #A8E90,
for #R3720.
3. 5:
1
and
3. 0:
1
for #J2F1, and 4.
3:
1
and 4. 1:1
This suggests that the parental AM- synthetase is more
sensitive to induction than that found in the mutants.
The rate at which extracellularly supplied methionine is
re-
moved from the medium by these organisms is shown in Figure
The most rapid accumulation is
carried out by strain #R3720. This
organism accumulated methionine at the rate of
dry weight equivalents /hour.
rate of
74
7.
mµmoles per mg
80
The parental strain was next with a
mµmoles /mg dry weight /hour. Strains #A8E90 and #J2F1
were the slowest accumulators, with a value of about
dry weight /hour. As seen in Figure
8,
64
mµmoles /mg
the intracellular level of
methionine remaining at each interval corresponded to the accumulation kinetics during the first two hours. After this time however,
the mutants can be seen to have entered an intracellular steady state,
considerably higher with strain #R3720, and somewhat higher with the
other two mutants, than was found with the parental strain.
This ob-
servation is partially explained when one examines the rate of disappearance of the internally accumulated methionine. This is shown in
Figure
9.
Although methionine is entering #R3720 at some
6
mµmoles /mg
dry weight /hour faster than it is into #3701B, it is disappearing at
the rate of 41 mµmoles /mg dry weight /hour, or
5
mµmoles /hour
slower than the wild -type. The disappearance of methionine can safely
29
80
70
m 60
o
3701B
A8E90
X
J2F1
A
R3720
E
T 50
4Q
v
m
E
o
40
m
ó
t
Z
20
10
1
2
3
4
Time (Hours)
Figure
7.
The accumulation of methionine during
incubation in the presence of 1mM
1- methionine.
30
3701B
° A8E90
J2F1
A R3720
X
2
1
3
4
Time (Hours)
Figure
8.
Intracellular methionine concentration during
incubation in
1
mM 1- methionine.
sO
ii
//
etlpnlne
elght
V1
nain
ge
Dry
g
O
ó
Enap
of
NO
neeples
ea.2
x
O
app
Dis
D
32
be assumed to represent conversion to adenosylmethionine (Pigg,
Sorsoli and Parks, 1964; Svihla and Schlenk, 1959). Although the
intracellular concentration of methionine in #R3720 is twice that of
the parental strain after two hours, the disappearance of methionine
in this mutant ceased at the same time as #3701B.
The rate of methi-
onine disappearance is slower for strain #J2F1 (30 mµmoles /hr) and
#Á8E90 (36 mµmoles /hour).
Because these latter two mutants do not
accumulate methionine as fast as the other organisms, their reduced
ability to convert methionine into adenosylmethionine does not become apparent before the second hour.
Comparison of in vivo cellular reactions with in vitro reactions
has proved of value to these studies.
These studies included the
assay of cell -free preparations for transmethylase, AM- synthetase,
and serine hydroxymethylase activity.
If one observes Figure 10, it
is seen that these enzymes occupy key positions among the terminal
reactions for methionine biosynthesis.
Studies were initially carried out with strain #3701B to ascer-
tain the levels of these enzymes in the parental organism in the
presence of the compounds involved in these interconversions. The
levels of serine hydroxymethylase activity after cultivation in the
non - supplemented medium, and after pulsed homocysteine plus
serine, pulsed methionine, and continuous methionine treatments
are shown in Figure
11.
It can be seen that the availability of methyl
Homocysteine
Adenosylmethionine
Serine
(Transmethylase)
(Serine Hydroxymethylase)
Adenosyl homocysteine
Methionine
X-C
Glycine
Jr
Homocysteine
+
Adeno sine
ATP
(AM- syntheta s e)
1
Adenosylmethionine (AM)
AM Pool
Figure
10.
Proposed methionine biosynthetic pathway in Saccharomyces
cerevisiae (Pigg, 1962).
34
a
.r.,
900
a
á,
800
.r.,
700
U
Qi
a)
a)
z
600
o
a)
o
500
E
rd ó
Gy
a) .,9
400
300
;-1
cu
up
(0
o
t
200
100
a)
a)
ÿ
O
z
.
a
N
0
o
CO
.
a)
p
0
0 °
.::01
rizy
ó
U
°'
x
Supplements
Figure
11.
Serine hydroxymethylase activity in
strain 3701B during exponential growth
in supplemented and non - supplemented
WCLM media.
35
groups from the serine methylated co- factor is significantly reduced
in the supplemented cultures over that of the non - supplemented media.
It can be seen in Figure 12, that transmethylase shows
ly the opposite effect.
precise-
Although it was expected that homocysteine
would stimulate further transmethylase synthesis, it was unusual to
find that methionine is even more effective in this respect.
thetase (Figure
13)
AM -syn-
shows the same stimulation with methionine. This
was expected from earlier results (Pigg, Sorsoli, and Parks, 1964).
It has now been demonstrated that homocysteine elicits a similar
response in the AM- synthetase.
When the wild -type is compared to the three mutant
strains of
this study after cultivation in media supplemented with the same
amino acids as above, one detects a variety of responses. With serine hydroxymethylase, shown in Figure 14, one observes that the
parental strain #3701B does not vary greatly from strains #J2F1 and
#A8E90 in non - supplemented and homocysteine plus serine supple-
mented WCLM media. When methionine is present, however, more
than twice the activity is discovered in the mutants. Strain #R3720
is quite different from the other organisms.
It shows only about one
half the activity of the other organisms in the non - supplemented
medium, a slightly higher activity than the others in homocysteine
plus serine supplemented medium, and approximately twice the ac-
tivity of #3701B, and slightly less than the other mutants in the
36
220
-
200
180
160
ú W
140
m
44
a)
.
120
100
w
El
0
?
o
U 80
60
40
20
2
co
á,,5,
a,
O
z
;-1
Ú
U)
O
a)
0b
X
Figure
12.
.
ó
U
a,
Methionine
cd
Supplements
Transmethylase activity in strain
3701B during exponential growth in
supplemented and non - supplemented
WCLM media.
37
2000
1800
4_;
.g
T
4-,
U
<4
a)
Q
1400
òA
d
1200
CO)
1'1
1600
4-9
a)
x .,
.4-,
1000
(i)
ó
800
U
600
400
200
a)
0
o
(1)
,
U "-i
Z
o
ch
(1)
0 0
0
0 o
U
U)
''a
Methionine
<4
Cf)
Continuous
Methionine
I
Supplements
Figure
13.
AM- Synthetase activity in strain
3701B during exponential growth in
supplemented and non - supplemented
WCLM media.
38
Supplements:
None
(3)
ó 1400
E
Homocysteine
and Serine
1300
Methionine
1200
C
.r.,
o 1100
g
;,
1000
o
Lv
á
900
.'.,
a)
u)
m
a)
800
ó
700
600
T
.,
4-3
U
500
Qi
a)
rn
ca
400
á
300
X
o
ro
200
-
>,
.,
100
3701B
Figure
14.
A8E90
Strain
J2F1
R3720
Serine hydroxymethylase activity in wild -type and mutant
strain after cultivation in supplemented and non supplemented WCLM media.
39
methionine supplemented medium.
The levels of transmethylase under the same conditions are
shown in Figure 15.
Strain #A8E90 reflects those levels found in the
parental strain, although not as high. Both #J2F1 and #R3720 show
greatly reduced levels of transmethylase in all media, and except for
some stimulation of #R3720 in homocysteine and serine, the trans-
methylase activity is not stimulated, even when methionine is present.
AM- synthetase assays in the same media are shown in Figure
16.
Unlike the parental strain, both strain #A8E90 and #R3720 show
lower levels of AM- synthetase when homocysteine and serine are
added to the medium.
Methionine stimulates this enzyme as in the
wild type; in #A8E90 to a lesser degree, and in #R3720 to a larger
degree. Although the level of AM- synthetase is considerably less
in #J2F1 in the non - supplemented medium, this organism shows
somewhat higher levels in the presence of methionine and homocys-
teine plus serine supplemented WCLM media.
An additional approach was
considered necessary in order to
determine the relative distribution of methionine and its methyl
group, and ethionine and its ethyl group, throughout the cellular
components during active growth in these compounds. All four or-
ganisms were tested in respect to the concentration of these compounds or their derivatives in the expandable pool, internal pool, hot
TCA- soluble fraction (containing the nucleic acids), the protein, and
40
Supplements:
None
Homocysteine /
and Serine
A
130
Methionine
120
110
100
..
.r.,
a)
y
90
o
H
a
4-)
bo
80
x
-5
70
c)
1
60
s,
m
E-1
1
44
U
50
0
40
1
30
20
10
1
3701B
Figure
15.
A8E90
Strain
J2F1
1
R3720
Transmethylase activity in wild -type and mutant strains
after cultivation in supplemented and non - supplemented
WCLM media.
S
41
upplements:
None
Homocysteine
and Serine
Methionine
3000
2800
.,,q
2600
-
2400
-
i
o
á 2200 2000
ñ
-
1800
0
o
U 1600
.,
1400
U
Qi
O
1200
y
-
1000
0
u)
800
<4
600
400
200
/i
i
/
A8E90 Strain J2F1
R3720
Figure 16. AM- synthetase activity in wild -type and mutant strains
after cultivation in supplemented WCLM media.
3701 B
42
the sterols. Homocysteine and serine were also tested to ascertain
the effect of these compounds on this distribution.
The accumulation of labelled methionine and ethionine into the
expandable pool is shown in Table
1.
The parental strain, #3701B is
not appreciably different for any of the media.
The amount of label
increases in this pool in the mutants, whether in methionine or ethionine, when homocysteine and serine are present in the medium.
The concentration of label in the internal pools is shown in
Table
2.
Although this pool was not assayed to determine the rela-
tive amounts of methionine (or ethionine) and adenosylmethionine (or
adenosylethionine), it can be assumed on the basis of experiments on
the accumulation of unlabelled methionine that it consists almost en-
tirely
of the sulfonium
derivatives. According to Kempner and Cowie
(1960), the only methionine present in this pool should be destined for
protein synthesis. The conversion of ethionine to adenosylethionine
has been described by Parks (1958a).
A
very important character shared by the mutants is shown by
observing the internal pool. From four to seven times as much of
the ethyl- sulfonium derivative is present in the wild -type as in the
mutants. Although all of the organisms show similar relationships
to each other when cultured in methionine supplemented media, the
presence of homocysteine and serine produces quite different results between the parental strain and mutants during incubation in
43
Table
1.
Concentration of Labelled Methionine and Ethionine in the
Expandable Pool. *
Supplements
Organisms
:*
#3701B
#A8E90
1- ethionine
0.44
0.43
0.30
0.41
1- ethionine
1- homocysteine
1- serine
0.41
1.
20
0.45
0. 57
1- methionine
0.41
0.52
0.33
0.89
1- methionine
1- homocysteine
1- serine
0.47
0.86
0.46
1.03
*
**
#J2F1
#R3720
14
Results expressed as mµmoles methyl -C or ethyl-C14/
mg dry weight cells
Ethionine and methionine were labelled ethyl-C14 and methyl C 14, respectively. Homocysteine was present as the thiolactone. Initial concentration of each supplement was 1mM.
44
Table
2.
Concentration of Labelled Compounds in the Internal
Pool after Incubation in Isotopic Methionine and
Ethionine. *
Organism
Supplement
1-ethionine
#J2F
1
#R3720
4. 13
7.15
3.97
23.60
8.80
8. 15
4.80
14. 50
6,70
7. 50
11. 70
26. 00
13. 40
10. 75
17. 00
#3701B
#A8E90
29.80
1- ethionine
-homocysteine
1- serine
1
1
-methionine
-methionine
-homocy steine
1- serine
1
1
*
14
14
Results expressed as mp.moles methyl-C14 or ethyl-C14/
mg dry weight cells.
Ethionine and methionine were labelled ethyl-C14 and
methyl-C14, respectively. Homocysteine was present as
the thiolactone. Initial concentration of each supplement
was 1 mM.
45
ethionine. The amount of label in this pool is reduced in #3701B when
the supplements to ethionine are present. It is increased in the mu-
tants under these conditions.
In the hot TCA- soluble fraction, shown on Table 3, the
pres-
ence of homocysteine and serine reduces the level of radioactivity in
all organisms when ethionine is present. As in the internal pool, the
presence of homocysteine and serine in methionine supplemented
media results in an increase in the label with all organisms. The
character
of methyl -group
transfer into nucleic acids has been de-
scribed by Borek and Srinivasan 1965).
(
The incorporation of methionine and ethionine into protein may
be seen on Table 4.
Homocysteine is observed to reduce the incor-
poration of ethionine in the wild -type strain, however no change can
be seen in the mutants.
Virtually no differences exist between the
organisms in the methionine supplemented media.
The incorporation of methyl and ethyl groups into the sterols
are shown on Table
5.
As would be expected, these figures
corre-
late closely with those of the internal pool. This would be expected
since ergosterol, one of the principal sterols, is methylated by
adenosylmethionine under normal conditions (Parks, 1958b).
In Table
6
one may see the total accumulation of labelled
methionine or ethionine in the presence and absence of homocysteine
and serine.
46
Table
3.
Concentration of Label in Hot TCA- soluble Fraction
After Incubation in Methionine -C 14H3 and EthionineCH2C 14H3*.
Supplement
Organism
.,,
#3701B
#A8E90
#J2F1
#R3720
1-ethionine
2.84
0.25
1.36
0.28
1- ethionine
1- homocysteine
1- serine
2.41
0. 12
0. 36
0. 24
1- methionine
4.70
3.25
2.88
3.42
1- methionine
1- homocysteine
1- serine
5. 60
3. 52
3. 66
3. 82
4Results expressed as mµmoles ethyl -C14 or methyl- C14 /mg
dry weight cells.
rJ
Ethionine and methionine were labelled methyl -C14 and ethylC14, respectively. L- homocysteine was present as the thiolactone. Initial concentration of each supplement was 1 mM.
47
Table 4. Incorporation of Labelled Methionine and Ethionine
into Protein.
Supplement
Organism
**
#3701B
#A8E90
#J2F1
#R3720
1-ethionine
1.32
0.46
0.76
0.59
1- ethionine
1- homocysteine
1- serine
0,90
0,44
0.80
0,57
1-methionine
21, 50
21, 70
23. 80
22, 20
-methionine
-homocysteine
1- serine
29. 10
22.20
29.60
24.20
1
1
*
**
14
Results are expressed as mµmoles ethyl -C14 or methyl -C14
incorporated /mg protein.
Ethionine and methionine were labelled ethyl-C14 and methyl 14, respectively. Homocysteine was present as the thiolactone. Initial concentration of each supplement was 1 mM.
48
Table
5.
Incorporation of Label from Methionine and Ethionine
into Sterols*
Organism
Supplement
#3701B
#A8E90
#J2F1
#R3720
1-ethionine
0. 56
1. 28
0, 71
0. 32
1-ethionine
1- homocysteine
0.40
0. 67
0.91
0. 63
1.49
1.
68
1. 37
2. 18
1. 87
1. 83
1. 59
1.92
1- serine
1
-methionine
1
-methionine
1- homocysteine
1- serine
Results expressed as mµmoles methyl-C14 or ethyl-C14/
mg dry weight cells,
**
Ethionine and methionine were labelled ethyl-C14 and methylC14, respectively. Homocysteine was present as the thiolactone. Initial concentration of each supplement was 1 mM.
49
Table
6.
Net Accumulation of Labelled Methionine and Ethionine
After Cultivation in the Amino Acid and Its Analog. '
Organism
J.J.
Supplement
#3701B
#A8E90
1-ethionine
34.
2
6.4
1- ethionine
1- homocysteine
1- serine
27.2
1- methionine
1
#J2F1
#R3720
9
5. 3
11.1
11.7
6,5
33.2
24.
1
24.5
29.2
48.9
31.
5
31.
35, 6
9.
-methionine
1- homocysteine
7
1- serine
Results expressed as mµmoles methyl -C or ethyl-C14/
mg dry weight cells. These figures are the calculated sum
to the measured pools in Tables -5.
Ethionine and methionine were labelled ethyl-C14 and methyl C14, respectively. Homocysteine was present as the thiolactone. Initial concentration of each supplement was mM.
1
1
50
DISCUSSION
The results demonstrate that the product of the AM- synthetase
reaction, adenosylmethionine (AM), does not limit the production of
the substrate methionine directly, but may slow its production by the
inhibition of serine hydroxymethylase. It is not known however,
whether the latter effect is due to AM or methionine.
When labelled adenosylhomocysteine (AH) was fed to the cul-
tures of yeast, it was found to be almost entirely converted to
AM
without significant dilution of the label (Duerre and Schlenk, 1962).
This strongly suggests that the direct conversion of AH to AM is of
great importance to the cell. Since the AM- synthetase enzyme is
maintained at a relatively high level, even in the absence of methionine, it would be reasonable to assume that the principal action of
this enzyme is in metabolite balance. By employment of the enzyme
in this manner, a nearly constant balance between methionine, AM
and AH could be maintained. In any event, one cannot conceive a
justification for the cells employing an additional mechanism to obtain the desired AM (at the expense of ATP) when the earlier reaction
would suffice quite well.
of
This is strongly suggested by the high level
serine hydroxymethylase enzyme found under these conditions.
Cultivation of the organism in homocysteine and serine or in
methionine support these conclusions. In both instances the
51
AM- synthetase and transmethylase levels increase, whereas the
serine hydroxymethylase is repressed. Cell growth is slightly inhibited with homocysteine and serine supplements and is strongly
inhibited in the presence of methionine.
This would suggest that the
increased consumption of ATP is sufficient to limit cellular processes. Since in the presence of methionine the transmethylase enzyme is also induced, it is logical to assume that the cell would
attempt to funnel the excess AM back into the terminal biosynthetic
system at this enzyme. If this were possible, a circle of ever faster
intermolecular conversions between methionine and AM would ensue;
the result being to squander vast amounts of ATP with no immediate
relief of the existing stress.
To avoid this eventuality, the
organism
apparently shunts the excess AM into the "non- recoverable" AM pool.
This pool has been described by Svihla and Shlenk (1960).
This would
reduce ATP consumption via the AM- synthetase to a minimum, and
the organism could be expected to return to a normal state soon after
methionine is removed.
Ethionine resistant mutants were prepared and tested under
the same conditions as the normal parental wild -type.
These were
found to be different from the mutants described by Sorsoli, Spence
and Parks (1964). In those mutants, a non -specific mutation resulting
in a loss of the ability to concentrate extracellularly supplied sub-
stances accounted for their analog resistance. Since those mutants
52
grow very slowly, one would not expect to obtain them by the isola-
tion technique employed in the current studies.
In these studies, no ethionine
resistant mutants were obtained
which showed increased levels of transmethylase.
Instead, all mu-
tants were found to have reduced levels of this enzyme, whether in
the presence or absence of methionine or homocysteine and serine.
If this enzyme were
repressible in some manner,
one would expect
that mutations to the constitutive state would be found. The advantages of this mutation are obvious. Still, it would seem imperative
that the cell be able to accumulate large amounts of methionine to
offset the analog effects. It can be seen from the results that this
does occur, not by mutation to a fully derepressed state, but by limiting the conversion of methionine to AM.
A
simultaneous loss of sus-
ceptibility to feedback inhibition, if this indeed occurs, would certainly benefit these mutants.
The enzymes have not yet been tested for
mutations of the latter type.
High levels of methionine were observed in the mutants in both
the absence and presence of homocysteine and serine.
of supplements did not
The presence
alter analog incorporation into protein as was
the case with the wild -type clone.
This would demonstrate that the
level of intracellular methionine in the mutants in the presence of
these supplements is not significantly different from that achieved in
their absence. Whole -cell accumulation data verifies this assumption.
53
When the AM- synthetase enzyme was assayed, it was found
that the levels of this enzyme in two of the mutants loosely corresponded to the rate of methionine conversion to AM as measured in
whole cells.
These rates were significantly retarded in these mutants
as were the levels of AM- synthetase. The third mutant, forming
about the same levels of the enzyme as the wild-type, converted in-
tracellular methionine to
AM much slower than the other
This would suggest alteration of the enzyme.
organisms.
This is further sup-
ported by data from ethionine accumulation experiments.
The cold
TCA- soluble fraction (assumed to consist principally of the sulfonium
derivative), represents
87
percent of the net accumulation in the
wild type. In the mutants, however, the same pool represents 65,
72
and
81
percent. With the exception of #J2F1, AM- synthetase
assays show that the pool size corresponds to the level of this enzyme.
The AM- synthetase enzyme again appears more selective in
mutant #J2F1 than is the case with the other mutants and the parental
strain. Inability of all mutants to accumulate large quantities
of
ethionine or adenosylethionine would suggest that some alteration
of this enzyme has
by data showing the
occurred in all the mutants. This is supported
larger size
of the AM pool found during incuba-
tion in methionine when compared to that measured after incubation
in ethionine.
In connection with discussion of this enzyme, the special case
54
of mutant #R3720 should be mentioned.
This organism is retarded
in its growth in non - supplemented media.
do not
Homocysteine and serine
alleviate this condition. Methionine, however, restored in the
organism to the growth rate of that of the wild -type organism the
.
non - supplemented medium. It was shown that this organism forms
the highest level of intracellular methionine. It does not, however,
display the characteristic "breaking point" concentration of the other
mutants, (i. e.
,
the point where the AM-synthetase reaches a level
sufficient to remove the methionine faster than it is accumulated).
It has also been shown that this organism has a low level of AM-
synthetase, and ceases converting methionine to AM when the intra-
cellular methionine level is still much higher than in the other organisms. These observations suggest that, in this mutant, a depletion of the AM occurs during cultivation in the non - supplemented
medium. This results for two reasons.
First,
AM is consumed in
the synthesis of methionine; and second, the organism cannot con-
vert the accumulated methionine back into
AM without the added
im-
petus of extracellularly supplied methionine.
Finally, results with the serine hydroxymethylase enzyme indicate that at least one site of control exists prior to the terminal
reactions. This effect is classical in nature. In the presence of
methionine the level of the enzyme was greatly reduced. Mutants
resistant
to ethionine showed much
less sensitivity to the effects of
55
methionine, producing up to approximately three times the amount of
the enzyme under these conditions.
This reaction alone could ef-
fectively control the intracellular concentrations of methionine and
AM.
From the preceding discussion, one may draw the following conclusions:
(1) In
addition to AM- synthetase, the transmethylase en-
zyme is induced in the presence of methionine or homocysteine plus
serine;
(2) the
serine hydroxymethylase is repressible by either
methionine or AM, or possibly both; (3) in non - supplemented media,
the balance of the terminal products is rather rigidly maintained by
intramolecular conversion between the terminal products;
(4) the
ultimate control of the quantitative level of the combined cyclic corn pounds is exerted somewhere outside the terminal cycle; (5)
resis-
tance to ethionine results from reduction in AM- synthetase activity
by reduction in the ability to form the enzyme, and possibly by al-
teration of specificity; and,
(6)
mutants may partially overcome
ethionine inhibition by relaxation of the repressive effect of methionine (or AM) on serine hydroxymethylase.
56
SUMMARY
In the presence of excess methionine both AM- synthetase and
transmethylase enzymes were induced.
A
similar, though reduced
effect was observed with homocysteine and serine supplements.
The serine hydroxymethylase enzyme was
repressed under these con-
ditions. Cellular regulation of the terminal cycle can only be conceived if one assumes that there is active intramolecular conversion
between the compounds involved. There would be no necessity for
repressive control
of these enzymes if one of the molecules entering
this cycle could be controlled. This requisite is filled by the serine
hydroxymethylase enzyme.
Support for the cyclic control phenomenon discussed was ob-
tained in studies with ethionine -resistant mutants of the parental
strain. These organisms did not overcome ethionine inhibition by
producing large quantities of transmethylase, but rather by a reduction in the level (and possibly by alteration of the specificity) of the
AM- synthetase enzyme.
The ability to synthesize AM- synthetase
was never completely lost by the mutants.
This would suggest that
complete disruption of the terminal cycle is not compatible with
survival.
57
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