Oxygen is not only one of the most abundant elements on the Earth, but it is also one of the most important elements for life. In terms of composition, the feature of the atmosphere that most distinguishes Earth from other planets is the presence of abundant amounts of oxygen. The first forms of life may have been similar to present day anaerobic bacteria such as clostridium. The relationship between prokaryotes and eukaryotes, if any, has been a topic of much speculation. With only a few exceptions eukaryotes are oxygen-utilizing organisms. This research eukaryotes or eukaryotic biochemical processes requiring oxygen, could have arisen quite early in evolution and utilized the small quantities of photocatalytically produced oxygen which are thought to have been present on the Earth prior to the evolution of massive amounts of photosynthetically-produced oxygen

Satyanarayana, T.

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Asaects and Rcie of Oxygen ire Eucaryat 1C Druanlsms	 4
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Carbon, hydrogen, oxygen and nitrogen are the four principal
elements that contribute to living matter. In catalysis and
energy exchange reactions iron and phosphorous are of critical
importance. Each of these elements is a common const ituent of
the earth and presumably formed during the formation of the
solar system. Carbon monoxide, car-Loon dioxide, water, methane,
amornia and hydrogen and oxygen in molecular form (H2 and 02)
are comre:^n molecules formed by the important elements. All of
these, except for oxygen have been considered to be the
constituents of the primitive atmosphere. Oxygen is not only
one of the most abundant elements on the earth, but it is also
one of the most important elements for life. Such being the
case, it has been the subject of intensive investigation among
a number of physiologists and biochemists since the study of
biological oxidation processes was initiated by Lavoister- about
200 years ago. In terms of composition, the feature of the
atmosphere that most distinguishes earth from ether planets is
the presence of abudant amounts of oxygen. If one understands
what determined the oxygen content of the atmosphere today, then
that will most likely unravel the history of the abundance of
atmospheric oxygen.
The upper- atmosphere of the earth is as dry as the Martian
atmosphere, and it has been proposed by Urey that the oxygen
on earth was produced by the photo dissociation of water.
Further studies by Urey have revealed that there would be
shielding of the main body of water vapor in the troposhere,
thus resulting in an auto-regulated level of oxygen produced in
a primitive atmosphere, a condition that is remarkable stable.
GBerkner and Marshall calculated this level to be about 1/1080th
or less of the oxygen of the present day atmosphere_
It is usually thought that the earth was once completely
anaerobic, containing w ­. ane and, possibly, More complicated
organic compounds. The first forms of life may have been similar
to present day anaerobic bacteria such as clostridium.
Evolution of the photosynthetic cleavage of water to oxygen
was doubtless a major event with far-reaching consequences.
Biologists generally believe that as oxygen accumulated in the
earth's atmosphere, the obligate anaerobes became limited to
strictly anaerobic environments. In the meantime, new classes
of bacteria appeared with microorganisms for detoxifying oxygen
and for using oxygen to obtain energy.
The relationship between prokaryotes and eukaryotes, if any,
has been a topic of much speculation. Eukaryotic cells contain
a variety of membraneous organelles, at least some of which are
probably the descendants of prokaryotic ancestors. The question
of the time of origin of the eukaryotes is of interest in
relation to NASA's program on the origin and early evolution of
life- With only a few exceptions eukaryotes are oxygen-utilizing
organisms. One question we would like to examine is whether
eukaryotes or eukaryotic biochemical processes requiring oxygen,
could have arisen quite early in evolution and utilized the small
\at
quantities of photocatalytically produced oxygen which are
thought to have beer, present on the earth prior to the evolution
of massive amounts of photosynthetically-produced oxygen. An
insight into the time of origin of the first eukaryotes may be
I
obtained by studying the cellular processes affected by oxygen.
One candidate for such a study would be the acetyl -coenzyme A
(ACS) synthetase of Saccharomyces cerevisiae.
The existence in the microbial world of organisms capable of
both fermentation and respiration, dependent on the oxygen level
of their environments, is of importance for the understanding of
the early evolution of life. Oxygen has a pronounced effect on
the growth, general metabolism and lipid composition of the
Eucaryote, yeast. The genus Saccharomyces has been used to
study the effects of oxygen on various physiological parameters
of yeast. These organisms provide several systems for study of
the role of oxygen in Eukaryotes. There is, however, a great
deal of uncertainty as to the precise role of oxygen in
determining the morphoiogical and biochemical changes that are
seen in aerobically metabolizing yeast cells. It is well known
that the synthesis of unsaturated fatty acids, sterols,
ubiquinone and a number of hemoproteins is inhibited by exluding
oxygen from the culture medium. , This is a consequence of the 	 ',,
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direct involvement of molecular oxygen in the biosynthesis of
the substances. However, other changes are known to occur in
these yeast cells as a consequence of non-aerobic environment,
for example, degradation of mitochondria, arid, in turn, enzymes
of tricarboxylic acid cycle.
Oxygen tension and glucose repression, regulate the formation
of mitochondria in yeast. One of the enzymes associated with
this organelle is acetyl-Coenzyme A synthetase (ACS), which is
the key enzyme concerned with lipid synthesis in S. cerev-isiae.
Earlier studies on this enyzme have revealed the presence of two
"N,
distinct acetyl-CoA synthetases dependent on the availability of
oxygen during growth of this organism. Under aerobic or oxygenic
conditions, the enzyme is associated with the mitochrondrial
fraction. Under non-aerobic conditions, it is associated with
the microsomal fractions of the cell homogenate. Further
studies, using isolated fractions revealed that the enzyme formed
during the two conditions of growth differed in a number of ways.
The affinity constant for acetate, was 10-fold higher and for-
ATP, 3-4 fold higher for the non-aerobic enzyme compared to the
aerobic variety of the enzyme. Long chain acyl-Coenzyme A
compounds (palmityl, stearyl and oleolyl) were found to be potent
inhibitors of ACS from aerobic, but not from non-aerobic cells.
The inhibitory pat!iern by these long chain acyl-CaA compounds
had characteristics of regulatory enzymes, with an interaction
co-efficient of 3.25. This suggested that the aerobic enzyme
is trimeric in structure. Short chain aryl-CaA compounds
(propionyl, butyryl and valeryl) had no effect on ACS from either
aerobic or non-aerobic source.
The "aerobic" ACS was purified to homogeneity. By various
criteria, the molecular weight has been deduced to be around
250,000. This study confirms the enzyme to be a trimer with a
subunit size around 83,000, this concurring with acyl-CoA
inhibition studies.
In further experiments, some of the immunologic properties of
the two ACS'S were studied in detail. The antibody produced by
each enzyme is immunologically specific. Antibodies prepared
from homologous enzyme inhibited only the homologous enzyme
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actifity but not the heterologous enzyme activity.
During these immunological studies, a partially purified non-
aerobic preparation of the enzyme has been used. This was
necessitated because attempts to purify this enzyme further were
hindered because of the labile nature of the enzyme during
purification procedures carried out at 4 aC. Indeed both the
enzymes were found to be unstable upon storage conditions at
4C C. However, addition of boiled extracts of aerobic, non-
aerobic or "petite" strains of the parent strain, prevented
such losses -f activity for both the aerobic and non-aerobic
RCS's. The instability of both the enzymes was shown not to
be due to the interference r=:f intracellular- proteinases since
specific inhibitors of these proteinases had no effect in
.i	 preventing ACS from inactivation. The stabilizing factor
r
(S.F.) present in the boiled extracts showed differential
effects during further characterization. The S.F. was non-
dialysable in the case of non-aerobic ACS stability while
it was partially dialysable with regards to aerobic ACS.
Charcoal treatment of the boiled extracts abolished the
stabilizing capacity toward bath the enzymes. Pronase
treatment destroyed the S.F. when tested against the non-
aerobic ACS but had no effect on the aerobic ACS. The S.F.
was partially purified on 0-100 Sephadex columns, and during
such studies the S.F. cochromatographed with cytochrome C.
indicating the molecular weight to be around 13,000. Further,
I
	
	
the S.F. showed a single absorption maximum at 260 nm. These
studies indicated the S.F. composition may be nucleopeptidic
While several nucleotides afforded protection to aerobic ACS,
only adenine derivatives, ATP and ADP afforded complete
protection at physiological concentrations. However, none of
these nucleotides showed any capacity to protect non-aerobic ACS.
Digestion of S.F. with either ribonuclease T, or
deoxyribonuclease did not eliminate the protective capacity
against either ACS. The ratio of the absorbance at 280 to
260 nm of S.F. coincided only with Poly G absorption
characteristics. However, synthetic Poly G afforded
protection only to aerobic ACS but inactivated non-aerobic ACS.
This property of Poly G could be abolished by digestion w4.th
ribonuclease T. Since the digestion studies with the
ribonuclease T, showed differential effects compared to S.F.,
the nucleotide portion of the S.F. may not be guanine
nucleotide derivative. This is in complete accord since only
adenine nucleotides were effective as indicated above.
Further prolification of the nucleopeptide on ion-exchange
	
a
4
columns completely dissociated the =60 rim absorbing material from
the peptide moiety. The nucleotide was identified as ATP both
by ATPase digestion studies and its substitution in the in vitro
assays replacing ATP. The peptide moiety was purified to
homogeneity as revealed by several physical studies. Its
apparent molecular weight was estimated to be around 13,000
500 by gel permeation chromatography. The stabilizing property
of the peptide was abolished completely by digestion, with
chymotoypsin only but not by any other proteolytic enzyme.
Using the purified peptide during the purification of
0?
the non-aerobic Acs, a 1300 fold purification was achieved.
On disc gel electrophoresis, the material revealed only two
bands. whether these two bands corresponded to the subunits
of the sxime enzyme could not be ascertained due to lack .)f
sufficient material.
Finally the existence of two isozymes was confirmed by
mixing experiments. Both the purified enzymes, when mixed,
could be separated as Bio gel - eolumns as well as dizc gel
electrophoresis. While [two project has contributed immersely
towards the understanding of the regulation and structure
aspects of the isozymes], the exact role of oxygen and the
evolutionary relationships to categorize the enzymes into
convergent or, divergent evolutionary pattern cannot be
ascertained at this stage of the project due to the
difficulty in obtaining sufficient amounts of proteins to do
the detailed structural analysis.
v
Y
1. Inhibition of Acetyl- Coenzyme
cer_ev_isiae by Long-Chain Acyl-
T. Satyanarayana and Hato 1 d P.
IVth International Fernientatio
Kyoto, Japan, April 1972.
Dr. T. Satyanarayana
A Synthetase of Saccharomyces
Coenzyme A Compounds.
Klein. Proceedings of the
n Symposium on Yeasts.
2. Inhibition of Acetyl- Coenzyme A Synthetase of Saccharcmyces
'cerev- isiae by Long-Chair, Acyl Coenzyme A Compounds.
T. Satyanar-ayana and Harold P. Klein. American Chemical
Society 164th National Meeting, August 27-Sept 1, New
York, (1972).
3. Isolation and Crystallization of Acetyl- Coenzyme A
Synthetase of Yeast. T. Satyanar^ayana and Harold P. Klein.
Pact. Proc., page 158, (1973).
4. Studies on Acetyl- Coenzyme A Synthetase of Yeast:
Inhibition by Long-Chain Acyl Coenzyme A Esters.
T. Satyanarayana and Harold P. Klein. J. Bact., 115,
600 (1973).
5. Studied on Acetyl- Coenzyme A Synthetase of Saccharomyces
cerev- isiae. T. Satyanarayana and Harold P. Klein.
Proceedings of the IVth International Symposium on Yeasts.
Vienna, Austria, A34, (1974).
6. Evidence for Two Immunologically Distinct Acetyl - Coenyzme
A Synthetases in Yeast. T. Satyanarayana and Harold P.
Klein. Biochern. Biophys.. Acta. , 341, 396 (1974).
7. Stability Characteristics of "Aerobic" Acetyl- Coenzyme A
Synthetase of Yeast. T. Satyanarayana and Harold P. Klein.
Fed. Pr•oc. , 35, 1668 (1976).
8. Studies on the "Aerobic" Acetyl- Coenzyme A Synthetase of
Sacchar_omyces cerev- isiae: Purification, Cr-ystallation
and Physical Properties of the Enzyme. T. Satyanarayana
and Harold P. Klein. Arch. Biochern. Biophys., 174,
480 (1976).
9. Comparative Enzymology of Two Distinct Acetyl- Coenzyme
A Synthetases of Sacchar_omycgs cerev-isiae.
T. Satyanarayana and Harold P. Klein. In IVth International
Fermentation Symposium on Yeasts. Montpellier, France,
July 1978.
10. Monoclonal Antibody as a Tool for- Further Differenciation
of the Two Acetyl- Coenzyme A Synthetases (ACS) of Yeast.
T. Satyanarayana, Harold P. Klein, V i r • g i n i a Johnson  and
Richard A. Goldsby. Fed. Proc., 39, 928 (1980).
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 1
i
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11. Subunit Structure and Immunological Distinctness of the
Two Acetyl- Coenzyme A Synthetases of Yeast.
T. Satyanarayana, Charles H. Chevenka and Harold P. Klein.
Biochem. Biophys. Acta., 614, 601 (1980).
12. Naturally Occuring Stabilizing Ligands as a Mode for
Further Differentiation of Two Acetyl- Coenzyme A
Synthetases (ACS) of Yeast. T. Satyanarayana and Harold
P. Klein. Amer. Soc. Microbiol. Meetings, K 20, (1981).
13. Rapid Purification of Fluoroscent Enzymes by Ultra-
Filtration. Morris A. Benzaminson and T. Satyanarayana.
Prep. Biochem. , 1, 24 (1983) .
14. The Relationship Between Aerobic Metabolism and }he
Acetyl- Coenzyme A Synthetase of Saccharomyces cerevisiae.
T. Satyanarayana and Harold P. Klein. West Coast Bacterial
Physiologists Annual Asilomar Conference, Pacific Grove,
California, December, 14-16, 1982.
15. Poly-Nucleotide Interactions with the Two Acetyl- Coenzyme
A Synthetases of Yeast. T. Satyanarayana and Harold P.
Klein (In Preparation).
16. Stability Studies of Isozymes: Part I. Localization of
the Stability Factors for the Isozymes of Acetyl- Coenzyme
A Synthetases of Saccharcmyces cerev-isiae.
T. Satyanarayana and Harold P. Klein (In Preparation))
17. Stability Studies of Isozymes: Part II. Identification
of the Stability Factor(s) for the Aerobic Acetyl- Coenzyme
A Synthetase of Sacrharomyces cerevisiae.
T. Satyanarayana and Harold P. Klein (In Preparation).
18. Stability Studies of Isozymes: Part III. Characteristics
of a naturally occuring Peptide Stabilizing Agent for the
Non-Aerobic Acetyl- Coenzyme A Synthetase of Saccharomyces
cerev- isiae. T. Satyanarayana (ln Preparation).
19. Rapid Separation of Interfacing Chromatofocusing Poly
Buffer from Peptide Samples of Similar Sizes.
T. Satyanarayana, Swedish Marketing Technica, Uppsala,
Sweden (1983).
20. LAB NOTES: Hydroxylapatite Facilities Rapid Removal of
Polybuffers from Peptide of Closely Related Molecular
Weights. T. Satyanarayana, Biologics., Calbiochem-
Behring Newsletter i0, 2 (1984).
21. Purification and Properties of Non-Aerobic Acetyl- CoA
Synthetase of Sacchar_omyces cerev-isiae.
T. Satyanarayana and Harold P. Klein (In Preparation).
jV
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22. Structure and Function of Isozymes in Saccharomyces
cer_ev_ i s i ae. T. Sat yanarayana and Harold P. Klein.
VIth International Symposium on Yeast;, July 9-13,
Montpellier, France, page VI-3-0 (1984).
00


Structure and function of isozymes: Evolutionary aspects and role of oxygen in eucaryotic organisms

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