MALEASE, A HYDRASE FROM CORN KERNELS

94 Pages·03.342 MB·English

by  SACKS, WILLIAM

DOCTORAL DISSERTATION SERIES
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DEGREE PUBLICATION NO
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The Pennsylvania State College
The Graduate School
Department of Agricultural and Biological Chemistry
MALEASE, A HYDRASE FRQI.i CORN KERNELS
A Thesis
by
Yrf'illiam Sacks
Submitted in partial fulfillment
of the requirements
for the degree of
DOCTOR OF PHILOSOPHY
June 195>1
Approved
PrpfesTsor of Phytochemistry
Head, Department of Agri-
cultural and Biological
Chemistry
TABLE OF CONTENTS
Page
I. INTRODUCTION ......................................... 1
II. HISTORICAL........................................... 3
A. The Szent-Gyorgi Cycle ....................... 3
B. The Tricarboxylic Acid (TCA) Cycle ......... 5
1. Kreb^ Citric Acid Cycle....... 5>
2. Individual Reactions in the TCA Cycle 8
a. The Formation of Isocitric and
Citric Acids .................. • 8
(1). The Condensation of
Oxaloacetic and Pyruvic
Acids (Krebs Conden­
sation) ................ 8
(2). The Condensation of
Oxaloacetic Acid with
Acetyl Phosphate . . . . 9
b. The Conversion of Citric Acid
and Isocitric Acid to Succinic
A c i d ..... 13
c. The Oxidation of Succinic Acid . 13a
d. The Conversion of Fumaric Acid
to Malic A c i d . 16
e. The Oxidation of Malic Acid . . 19
(1). The "Malic" Enzyme . . . 19
3. The Coupling of Phosphorylation with
Oxidation..... 20
Page
l+. Carbon Dioxide Fixation................ 22
5. Other Enzymic Reaction Involving
TCA Cycle Acids......................... 214.
a. Decarboxylation of TCA
Cycle Acids..........................27
6. The Hew TCA Cycle..........................28
7. The Cyclophorase System...................30
8. Site of the Oxidative System in the
C e l l ....................................... 31
9. The TCA Cycle in Plants 31
III. EXPERIMENTAL............................................33
A. Apparatus.........................................33
B. R e a g e n t s .......... 33
C. Preparation of ’’Malease’1 ..............31+
D. Test of ’’Malease” A c t i v i t y .................... 3&
S. ''Malease” Activity Determined by Polaro-
graphic Estimation of Maleic A c i d ........... 37
F. Formation of Malic Acid from Maleic Acid
by "Malease” .....................................1+8
G. Test for Fumaric Acid in the Conversion
of Maleic A c i d ...................................50
H. Formation of Maleic Acid from Malic Acid
by "Malease" . • .............. • • • • • • • 52
I. Test for Fumaric Acid in the Conversion
of Malic Acid ...................................51+
J. Formation of Maleic Acid from Succinic Acid , 51+
Page
K« Polarographic Study of Oxaloacetic end
Pyruvic Acids................................. 56
L» Mixtures of Maleic and Pyruvic Acids . . . • 61)
M* Attempts to Identify Pyruvic and Oxaloacetic
Acids in Digestion Mixtures 67
IV, DISCUSSION...............................................68
V. SUMMARY ................. 7k
VI, BIBLIOGRAPHY............................................ 75
1
AC KNOWLEDGEI.IENTS
The author wishes to express his crateful appreciation
to:
Dr. C. 0. Jensen for his aid in planning the invest­
igation and for his helpful advice and criticism during the
preparation of this manuscript;
Rita and Harriet Lou for their help in preparing the
first draft of this manuscript;
Dr. P. J. Elving and Mr. I. Rosenthal for their
technical advice concerning the polarographic analyses.
i
I INTRODUCTION
The aerobic formation of succinate from fumarate, in
the absence of added pyruvate, was demonstrated by Krebs
and Eggleston (ij.6). Similarly, Lakl (£l) was able to
obtain succinate from maleate and reported the redox
potential of the succinate/maleate system as -0.091J. volt.
Dakln (17) could find no optically active malic acid
after digesting a 1% solution of neutral sodium maleate with
100 g. of fresh muscle, and noted that Hmuch unchanged
maleic acid was left in the solution and could be recovered
with ether.M
Contrary to the statements in the literature, Green
(28), in studying the effects of various reagents on the
oxidation of malate, found that maleic acid does not act
as an inhibitor. Also, he stated that in the presence of
unpurified maleic acid (commercial preparations) his
dehydrogenase system reacted vigorously with oxygen. However,
he believed this was due to traces of fumaric acid in the
preparation. When he added freshly distilled maleic
anhydride he reported that the enzyme system was "practically
inactive." This failure to get completely negative results
he disposed of by saying that "the conversion of maleic to
fumaric acid occurs to some extent at 37°*,f Since even a
partial conversion seems to require rather rigorous treatment
(i.e. treatment of a suspension of maleic acid in manganese
2
dioxide with sulfur dioxide (71) or heating to 99*9° in the
presence of HC1, IIBr, H2SO^, HCIO^, KCNS, NH^CNS, or NaBr
as catalysts (7U-)) It did not seem likely that a conversion
of maleic acid to fumaric acid actually did take place.
In general, previous investigations of the action of
enzymes catalyzing the loss of water by malic acid employed
methods of estimation of the end products which did not
differentiate maleic acid from fumaric acid (i.e. titration
with potassium permanganate or spectrophotometric measurement
of absorption in the ultraviolet). Since the work of Dakin,
better methods for the identification and estimation of
maleic acid and fumaric acid in the presence of both have
been published by Warshowsky and Rice (118) and Warshowsky,
Elving, and Mandel (117)• These authors used polarographic
measurements to identify and estimate these isomers.
After a consideration of the evidence discussed above,
we undertook an investigation of the enzymic conversion of
maleic acid using a polarographic method of identifying and
estimating maleic and fumaric acids. Since our enzyme
preparations contained malic dehydrogenase, the reaction
was driven in the direction of oxaloacetic acid by employing
triphenyltetrazolium chloride (TTC) as an electron
acceptor (38).
3
II HISTORICAL
A. The Szent-Gyorgi Cycle.
In 1931+ Gozsy and Szent-Gyorgi (26) found that the
addition of small amounts of fumaric acid to suspensions of
pigeon breast muscle greatly increased the rate of respi­
ration. A small amount of fumarate stimulated a comparatively
large amount of respiration and still was not used up* Also,
they noticed that small amounts of malonic acid added to the
tissue suspensions decreased the rate of respiration
considerably. In the presence of malonate, the addition
of fumarate restored the respiration rate.
Szent-Gyorgi and his coworkers (3» ij-)# in 1935 and 1936,
published a series of papers in which these observations
were confirmed and extended. At that time they stated that
the added fumarate merely maintained the respiration of
suspended tissues. Also, they reported that in the presence
of malonic acid, the fumaric acid which was added to the
tissue suspensions was "reduced” to succinate. As a result
of these and other observations, Szent-Gyorgi (108) presented
a theory for the oxidation of such substances as phospho-
glyceric aldehyde. This scheme was given as follows:
k
Donator
I
Activator Co-deh
I
- COOH
Malico H
Dehydr. OH
1
- C - COOH
Fumarase
1
Co-doh H
I
Flavo Prot
I "
- C - COOH
Succino | H
Dehydr. | H
- C - COOH
H
Cytochrom
A * B * C
Cytochrom Oxydase
In this scheme, the H of the donator, activated by its
dehydrogenase, was transferred on to oxaloacetate which was
activated by the malic dehydrogenase. From the malate the
H was transferred to coenzyme, from the coenzyme to diaphorase,
from diaphorase to fumarate which in turn was activated by