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Exchange in vitro of Subunits between Enzymes from Different Organisms Chimeras of Enzymes.

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E
Volume15
-
Number4
April 1976
Pages 181- 250
International Edition in English
Exchange in uitvo of Subunits between Enzymes from Different
Organisms: Chimeras of Enzymes
By Guido R. Hartmann[*]
Dedicated to Professor Feodor Lynen on the occasion of his 65th birthday
Most intracellular enzymes are made up of several identical or different subunits. The more
remote any two organisms are phylogenetically, the greater will be the differences in amino
acid sequence of a g/ven enzyme. Nevertheless, numerous examples exist of specific association
between chemically different subunits, or even of the formation of enzyme chimeras. They
span not only the boundaries between related organisms but also the deep rift between prokaryotic
and eukaryotic cells. Exchange of subunits between enzymes of similar activity but differing
origin can be rationalized by assuming enzymes to possess functionally defined types of threedimensional structures.
1. Introduction
The amino acid sequence of proteins is extremely varied.
Even enzymes of identical catalytic activity have far from
identical amino acid compositions in different organisms. An
example that has been studied particularly thoroughly is
cytochrome c isolated from plants and from lower as well
as higher animals; the length and amino acid sequence of
the polypeptide chain has ben determined. Notwithstanding
identical biological function, significant differences are found
in the length of the protein chain and quite considerable
variations in the sequence of amino acids. The differences
are all the greater the farther the organisms under study
are separated phylogenetically[']. However, this sets no limits
as to the polymorphism of enzymes, the occurrence of isoenzymes showing that even in a single organism several forms
of a protein may be present having the same catalytic specificity
in spite of differences in molecular structure['!
[*I
Prof. Dr. G. R. Hartmann
Chemisches Laboratorium der Ludwig-Maximilians-Universitat
Miinchen
lnstitut fur Biochemie
Karlstrasse 23, 8000 Munchen 2 (Germany)
Anyew Chrm. I m . Ed. Engl.
/ 1'01. 1 5 ( l Y 7 6 ) N o . 4
Under natural conditions, the amino acid sequence of a
polypeptide chain also determines the biologically active threedimensional structure : enzymes which have somehow lost
their three-dimensional structure without damage to the polypeptide chain spontaneously refold, albeit at different rates,
to their original shapec3'.In keeping with the enormous variety
of amino acid sequences in polypeptide chains there should
be just as many protein three-dimensional structures in nature.
Most intracellular enzymes contain more than one polypeptide chain[41. One distinguishes between homopolymeric
enzymes, made up of a frequently even number of identical
subunits and heteropolymeric enzymes composed of different
subunits. The isolated subunits are usually, but not always,
inactive.
The binding between the subunits in the complete and
active enzyme is mediated by highly specific interactions determined by the amino acid sequences of the subunits, for enzymes
consisting of subunits also spontaneously renature to the
biologically active quaternary structure after complete unfolding of the polypeptide chains15].The high specificity of association between the subunits of an enzyme is demonstrated by
the regeneration of the original activities of all the individual
enzymes on renaturation of a mixture of various dissociated
181
Yolvmeric enzymes[61 Analysis of normal and of pathological
hemoglobins has revealed that association to the active quaternary structure requires perfectly fitting areas of contact between the subunits. Even the slightest change, such as replacement of a single amino acid, can have an interfering effect"].
In the light of these considerations one would hardly expect
the subunits of oligomeric enzymes to combine in disregard
of barriers between species to give catalytically active hybrid
structures. The frequently considerable differences between
the amino acid sequences of proteins serving the same function
in different organisms should not permit association. Surprisingly, however, several experimental investigations have shown
that such enzyme associates, named chimeras after a fabulous
creature of antiquity (cf. Homer, Iliad, 6th Song, Verse 181),
can nevertheless be formed in uitro and even in uivo from
subunits arising from different species.
2. Hybrids of Homopolymeric Enzymes
2.1. Hybrids of Dimeric Enzymes
An example of intergeneric hybrid formation (by exchange
of subunits) has long been known to occur in the case of
alkaline phosphatase (EC 3.1.3.1) from bacteria. Under certain
conditions, this enzyme exists as a dimer of two identical
subunits. It has been isolated from both Escherichia coli and
Serratia marcescens. The two proteins are chemically very
different. Thus the peptide patterns obtained on tryptic degradation are not the same; and an antiserum produced by
the phosphatase from Escherichia coli hardly reacts with the
enzyme from Serratia marcescens.
If the two enzymes are dissociated into their subunits by
treatment with 6 M urea in the presence of thioglycolic acid,
the two solutions combined, and the action of the denaturing
agent stopped by dilution, then a considerable proportion
of the enzymatic activity is regenerated during several hours'
incubation at 37 "C. Electrophoretic analysis reveals not only
the bands of the original enzymes characterized by their various migration rates but protein exhibiting an electrophoretic
mobility intermediate between those of the starting enzymes.
We know from numerous studies on isoenzymes['I that the
appearance of proteins with modified electrophoretic mobility
under these conditions is indicative of hybrid formation, i. e.
generation of associates of subunits of the two original
enzymes. Leuinthal et al.['] justifiably conclude that in this
experiment too, in which enzymes from different species were
used, a hybrid consisting of one subunit 'U of the Escherichia
coli enzyme and one subunit 'U of the Serratia marcescens
enzyme is formed according to equation (1).
W),+ ("U),
+ 2(eUSU)
It would seem that protein-protein interactions occur
between the heterologous subunits which resemble those
between the autologous subunits[']. The hybrid protein isolated by electrophoresis displays phosphatase activity. However, this is not necessarily due to the hybrid itself, but could
possibly arise from monomeric subunits or parent enzymes
that may be present in a dissociative equilibrium (2)
182
2('USU)+ 2'U
+2"s
(eU),
+ ("U),
(2)
under the conditions of activity assay. In this context, the
observation that the equilibrium ( 2 ) can be shifted in either
direction by foreign molecules warrants
2.2. Hybrids of Tetrameric Enzymes
On reaction between the subunits U of tetrameric homopolymeric enzymes from two organisms a and b several hybrids
can be formed. Just on the basis of their stoichiometric composition, three hybrids (aUbU3; aU,bU, ; aU3bU)are possible.
But for any given composition various spatial arrangements
(planar or tetrahedral) are feasible in the associate. Since
the equilibria between the various spatial structures may differ
from those of the parent enzymes even more molecular species
can occur under certain circumstances[101.The existence of
the many conceivable interspecies hybrids will be decided
not only by thermodynamic aspects but also by the kinetic
factors of formation and decomposition[' 'I. Hence the number
of hybrids observed will differ from case to case.
Examples of chimera formation in enzymes composed of
four identical subunits have been observed for the Schiff-baseforming aldolase (EC 4.1.2.13)[12 15], L-lactate dehydrogenase
(EC 1.1.I .27)[16- 'I, and D-glycerinaldehyde-3-phosphate
dehydrogenase (EC 1.2.1.12)['1~19-231.
Formation of interspecies hybrids in these enzymes is favored by their ready dissociation under relatively mild conditions. Accordingly, effectors
which promote dissociation of the enzymes into their subunits,
such as ATP in the case of ~-glycerinaldehyde-3-phosphate
dehydrogenase[221,greatly accelerate hybrid formation.
It is found that the phylogenetic separation between the
hybridizing subunits can be very large and embraces the whole
range of eukaryotes ( e . g .beef x rabbit['31,trout x rabbit["],
Drosophila x rabbitLt4],roundworm x rabbit[15,"1, yeast
x rabbit[19x20.231).In a number of cases the associated polypeptide chains differ so greatly from each other that they
no longer show immunological cross-reactions. Evidence for
hybrid formation usually follows from the difference in electrophoretic mobility between the products and the starting
enzymes. Strictly speaking this merely indicates association
of the subunits of different origin. Whether, and to what
extent, the observed enzymatic activity is attributable to the
hybrids cannot be said for the reasons stated above for the
intergeneric hybrid of alkaline phosphatase (Section 2.1).
~
2.3. Hybrids between Enzymes of Related Activity
A particularly remarkable case of chimera formation is
that between the subunits of creatine kinase (EC 2.7.3.2) from
rabbit brain and L-arginine kinase (EC 2.7.3.3) of the sea
cucumber. These are two enzymes which differ not only in
origin but also, in spite of any similarity in the reactions
catalyzed, in their substrate. They both consist of two subunits
having relative molecular masses of about 40000. After dissociation of a mixture of the two enzymes into the subunits
with urea, analysis of the proteins present after reactivation
shows the presence of a new protein whose rate of electrophoretic migration lies between the values of the original enzymes[241.
This species is most probably the hybrid enzyme, for reaction
of both creatine and arginine to form the corresponding phosphorylated products according to equation (3) is catalyzed
Anyaw. Chem. I n t . Ed. Enyl. i Vol. 15 ( 1 9 7 6 ) N o . 4
in this protein zone, whereas each of the starting enzymes
can only react with one of the two substrates. However, for
the same reasons as were cited for the chimeras of phosphatase
the conversion of both substrates in this protein zone is not
necessarily due to the hybrid itself.
NH
II
R-CH,-N-C-NH,
I
+ ATP
--+
NH
II
R-CH,-N-C-NH-PO,H,
+
I
R’
ADP
R’
(3)
R = COOo, R‘ = CH,: C r e a t i n e
H = (CH,),-CH(NH,)-COOo,R’ = H: A r g i n i n e
Hybridizations between subunits of enzymes displaying different specificity even appear to occur in uiuo. Thus apart
from a dehydrogenase (EC 1.2.1.24), converting succinic
HOOC-CHz-CH2-CHO
+
N A D + + HZO
(4)
HOOC-CII,-CHz-COOH
+ NADH + H +
semialdehyde with the aid of NAD’ into succinic acid according to eq. (4), and a chromatographically distinct dehydrogenase (EC 1.2.1.19), transforming 4-aminobutanal with
NAD into y-aminobutyric acid according to eq. (5), two
further dehydrogenases have been isolated from a Pseudomonas
+
H,N-CH,-CH,-CH,-CHO
+
NAD+ + H
,
O
+ NADH + Hi
strain which can each react with both substrates. Judging
by the relative molecular masses, their composition of different
subunits, and their antigenic properties, the two last-mentioned
species are hybrids of the two starting enzymes[251.Remarkably, the subunits of the two parent enzymes associate with
the same stoichiometry in the hybrids in spite of the considerable difference of about 20000 in their relative molecular
masses.
3. Hybrids of Heteropolymeric Enzymes
3.1. Hybrids of Enzymes with Two Different Subunits
For the reasons discussed in Section 2.1 it is very difiicult
to establish in the case of hybrids of homopolymeric enzymes
whether the enzyme activity observed in electrophoretically
isolated proteins is really attributable to the chimeras. However such proof is required to demonstrate that association
of the heterologous subunits is as specific as that of the autologous subunits in the active parent enzymes. This question
can be answered much more easily with hybrids of enzymes
made up of different subunits (say cx and P), for in such
a case the isolated subunits cx and p have no activity of
their own, or one differing from that of the intact complex
(ap). Now if a mixture of subunit
from species a with
subunit ’P from species b displays the characteristic activity
of the complete parent enzyme (up) it is established that
the interactions between the heterologous subunits in the
enzyme chimera (“gbp) correspond to those in the parent
enzymes ( “ d p ) and ( b ~ b pin
) , as far as is necessary for establishment of catalytic activity.
A t i y e w C‘heni.
In[. E d . Enyl.
Vol. 15 ( 1 9 7 6 ) NO.4
it does not always follow that
~
(5)
H,N-CH,-CH,-CH,-COOH
Bacterial tryptophan synthetase (EC 4.2.1.20) consists of
two subunits of different size which can readily be separated.
Unlike the complete enzyme the isolated subunits, although
enzymatically active, are incapable of catalyzing the reaction
of indole 3-glycerophosphate with serine to give tryptophan
and D-glycerinaldehyde 3-phosphate. It has been possible to
combine the two subunits from a series of Enterobacteriacae
(e.g. Escherichin coli, Salmonella dysenteriae, Enturobucter
aerogenes, Serratia marcescens) over the species boundaries, in
spite of considerable differences in amino acid sequence, to
form hybrids exhibiting the activity of the complete parent
enzymes[26-281.In some cases it even proved possible to
form hybrids from subunits of enzymes from gram negative
and gram positive microorganism^[^^^ 301, and even of enzymes
from bacteria and blue-green algaeC3’].
Active enzyme hybrids could also be obtained from bacterial
anthranilate synthetase complex (EC 4.1.3.27) which is likewise
composed of two different subunits (cx and p). However, not
every combination of a subunit from a bacterium a with
a subunit from another bacterium b leads to an active hybrid
[equations (6a) and (6b)l. If
In these experiments subunits of enzymes from such widely
differing classes of Eubacteria as Bacillus, Pseudomonas, Acinetobacter, and Enterobacter were incubated togetherc3’ 361.
That enzymatic activity does not always appear demonstrates
that biological diversification can indeed also lead to modifications of the regions of the polypeptide chains of importance
for interactions between the subunits.
Chimeras can even be prepared in uitro from bacterial protein complexes in which one subunit catalyzes the entire reaction while the other subunit merely carries the binding site
of an allosteric effector; like the parent enzymes these chimeras
are also subject to allosteric regulation. This is demonstrated
by the formation of two active hybrids of aspartate transcarbamylase (EC 2.1.3.2) with allosteric control from the isolated
subunits of Escherichia coli and Sulmonellu typhimuriumt3‘I.
To the above examples from the field of prokaryotes one
can add the chimeras of heteropolymeric enzymes from eukaryotes. Thus the cx- and P-polypeptide chains of hemoglobin
from such different species as elephant, ass, mouse, and frog
can associate with each other to form hybrids[381,which in
all cases examined so far display the characteristic protondependent oxygen uptake of the complete tetramer’391.
~
3.2. Hybrids of Enzymes Having Several Different Subunits
3.2.1. Hybrids of DNA-Dependent RNA Polymerase
We may expect a precisely interlocking shape extending
over particularly large regions, and thus very narrowly limited
and specific interactions between the subunits, in those proteins
which are made up of several different polypeptide chains
and d o not dissociate under the conditions of a biological
environment.
183
Such an enzyme is bacterial DNA-dependent RNA polymerase (EC 2.7.7.6). It catalyzes the synthesis of RNA from ribonucleoside triphosphates under the control of DNA acting as
template. The enzyme is usually isolated from bacteria as
a complex of four different protein subunits a, p, p’, and
CT having the stoichiometric composition a2PP’o. Only the
o subunit can sometimes be removed from the complex by
ion-exchange chromatography without resorting to more drastic measures. The core enzyme having the stoichiometric composition a2pp’, however, is so stable that it dissociates only
in detergents such as dodecyl sulfate or in 6~ urea. None
of the isolated subunits displays any enzymatic activity. For
this reason it cannot be established whether the function
of the individual subunits in the catalytically active intact
complex changes from species to species. Comparison of the
RNA polymerase from Escherichia coli with that from various
bacteriophages or from mitochondria, which contains just
a single polypeptide ~ h a i n [ ~ ’ - shows
~ ~ ] , that DNA-dependent
RNA polymerase is certainly not identical, or even similar,
in all organisms. And even among bacteria, significant differences in size (Table 1) and charge are o b ~ e r v e d [between
~~,~~~
subunits from different species. Bacterial RNA polymerases
are also very different in their antigenic properties. For
instance, neither the enzyme from Escherichia coli reacts with
the antibody active against the enzyme from Micrococcus
luteus, nor does the Micrococcus enzyme with the antibody
active against the Coli enzymer4’
Table 1. Relative molecular masses of the subunits of RNA polymerase
from Escherichia coli and Micrococcus luteus [45].
Subunit
E. coli
M . luteus
Smallest subunit (a)
Medium-sized subunit (0)
Second-largest subunit (p)
Largest subunit
39 ooo
95 000
155000
165000
MOO0
80000
146000
151000
(v)
Nevertheless, comparison of physical data does not permit
any comparison of the functions of the subunits. In such
cases, functional equivalence can only be proved by mutual
exchange of the subunits.
Such an exchange involving two bacteria, which differ considerably on the basis of their DNA composition and all other
taxonomic criteria, uiz. the gram-negative Escherichia coli and
the gram-positive Micrococcus luteus, has been studied in
detail. It proved possible to replace the subunits CI, p’, and
o in the RNA polymerase from Escherichia coli by the similar
but different-sized subunits of the Micrococcus enzyme; DNAdependent RNA synthesis was observed with these enzyme
chimeras. Similarly, replacement of the smallest and the
second-largest subunit in the Micrococcus luteus polymerase
by subunits of the Escherichia coli enzyme also leads to active
enzyme chimeras (Table 2)[4s,461.-Attempts to obtain active
hybrids containing the largest subunit from Escherichia coli
and the second-largest from Micrococcus luteus have so far
failed.
The resulting chimeras display differing activities, based
on the amount of protein employed. It has yet to be established
whether the low activity registered in some cases is attributable to an unfavorable association equilibrium between the
subunits or whether the hybrid complex formed possesses
a low specific activity. As in all other cases of intergeneric
184
enzyme hybrids, precise characterization still has to be performed, especially with regard to physicochemical properties.
During the catalysis of RNA synthesis the various subunits
must fulfill corresponding functions in the two bacteria. Otherwise it would be impossible to rationalize the observation
Table 2. Exchange ofsubunits between the RNA polymerases from Escherichia
coli and Micrococcus luteus [45, 461. +, formation of a catalytically active
chimera; -, no detectable enzymatic activity.
____
-
Subunit
exchanged
-______
in E. coli
RNA polymerase
in M . luteus
RNA polymerase
+
+
+
____
Smallest subunit (a)
Medium-sized subunit (0)
Second-largest subunit (0)
Largest subunit (8’)
++
-___~
-
that each subunit can be replaced by a corresponding subunit
from the other bacterium in at least one of the two enzymes.
Moreover, any subunit can replace only one subunit in the
polymerase of the other bacterium. Thus an unequivocal functional assignment of the subunits of the two bacteria is possible.
At no stage of the divergent development of the two bacterial
strains has a transfer of functional purposes in RNA synthesis
taken place between one polymerase subunit and another.
The feasibility of exchange of subunits between various
species of bacteria provides a strong indication that not only
the function, but also the shape required for specific mutual
association of the subunits, is very similar. Otherwise the
formation of functioning enzyme chimeras would hardly be
understandable. The similarity between the corresponding
subunits from different bacteria is so great that mutations
in the subunits can be effective beyond taxonomic boundaries.
For example, a mutation in the gene of the P-subunit of
RNA polymerase from Escherichia coli has the result that
this enzyme is no longer inhibited by the antibiotic rifampiAn incorporation of the mutated subunit into the
rifampicin-sensitive RNA polymerase from Micrococcus luteus
yields an enzyme chimera that is resistant to the
Hybridization of the core complex azPp’ with the subunit
o has been accomplished with such different bacteria as Escherichia coli, Bacillus subtilis, Bacillus megatherium, Bacillus
cereus, Azotobacter uinelandii, Pseudomonas aeruginosa, and
Micrococcus I u t e ~ s 46,48
[ ~ ~ ,- 521. Success of the experiment can
be easily deduced from the appearance of enzymatic activity
characteristic of the complete enzyme azpp’o. A protein has
even been discovered in the chloroplasts of the single-cell
alga Chlamydomonas reinhardtii which acts as the o subunit
on the core enzyme a2Pp)of RNA polymerase from Escherichia
c0~i[531.
Reaction ofthesubunit o of a bacterium a with the heterologous core enzyme of another bacterium b, does not always
imply that the o subunit of bacterium b will form an active
enzyme chimera with the core enzyme of a. This observation
can be utilized to test which subunit in the core enzyme
a2PP’ is particularly important for the action of CT.Accordingly,
the p’ subunit is important for the function of o in Escherichia
coli, since o from Escherichia coli can only complement the
core enzyme azpp’ to form the active polymerase when an
RNA polymerase hybrid contains the p’ subunit from EscheriAll these results support the concept of a fundachia c0Ii[~~1.
mentally similar kind of organization of the RNA synthesis
system in .bacteria.
Angew. Chem. Int. Ed. Engl.
Vol. 1 5 (1976) No. 4
3.2.2. Hybrids of Bacterial Ribosomes
Similar observations as for RNA polymerase have been
made on ribosomes from bacteria. Ribosomes catalyze the
synthesis of proteins from activated amino acids on mRNA
with participation of several “soluble” proteins. Ribosomes
are made up of a larger and a smaller subunit, which are
in turn made up of RNA and 34 and 20 polypeptides, respectively. It had already been observed in 1966 that the two ribosomal subunits from gram-positive Bacillus subtilis and gramnegative Escherichia coli can be cros~-hybridized[~~!
The functional equivalence of the subunits and the contact surfaces
necessary for catalytically active interactions must therefore
have been retained during the evolution of ribosome structure.
The functional similarity even extends as far as the individual
proteins, for it is possible to replace almost every ribosomal
Escherichia coli protein by an equivalent protein from Bacillus
stearothermophilus, during reconstitution of the smaller ribosomal subunit from the isolated proteins and RNA, although
some of the Bacillus stearothermophilus proteins differ considerably from the corresponding Escherichia coli proteins with
regard to amino acid composition, electrophoretic behavior,
and immunochemical criteriafss1.
Some ribosomal proteins are responsible for species-specific
peculiarities as are observed, e.g., in protein synthesis on
the RNA of bacteriophage R 17. Such peculiarities also occur
in the chimeras obtained on incorporation of these protein
in heterologous ribosomesfS6~
571. Furthermore, not only can
ribosomal proteins be exchanged, but also ribosomal RNA
between the ribosomes of different bacteriafs8!
far the protein subunits have retained their function and possibly even their ability to engage in functional cooperation
across the frontier between prokaryotes and eukaryotes. Associate formation was observed between the subunits of 3-glycerinaldehyde 3-phosphate dehydrogenase from Escherichia coli
and rabbit or pig[231.The same has been reported for the
subunits of triose phosphate isomerase (EC 5.3.1.1) from
Bacillus stearothermophilus and chickenrz3! Initial studies on
ribosomes are now also available. The ribosome subunits
of eukaryotes are significantly larger than the corresponding
subunits from bacteria. Nevertheless, the small ribosome
subunit of the shrimp Arternia salina, a eukaryote, can cooperate with the large ribosome subunit from Escherichia coli
in an albeit restricted functional interaction (i. e. only in the
catalysis of polyuridylic-dependent dipeptide synthesis). The
physicochemicalinteractions between subunits displaying such
far-reaching phylogenetic differences are very weak. In order
to detect their association glutardialdehyde was added as
cross-linking agent during dipeptide synthesis; the hybrid ribosomes thus stabilized by covalent bridging could be detected
In
by the sedimentation velocities in the
another case, cooperation of the small eukaryotic ribosome
subunit from Chlorella with the large subunit from bacteria
could be established by ribosome-dependent synthesis of
guanosine tetra- and penta-pho~phate[~’].
Finally, it also proved possible to remove two acidic proteins
from eukaryotic ribosomes of yeast and functionally replace
them by two ribosomal proteins from Escherichia ~ o l i [ ~ ~ ] .
In this case, however, there exists an immunologically
demonstrable relationship‘’ ‘1.
3.2.3. Hybridizations with “Soluble” Protein Factors
Apart from ribosomes, “soluble” protein factors affecting
the processes occurring at the ribosomes participate in protein
synthesis. “Soluble” proteins from various bacteria were found
to be active on heterologous ribosomes in spite of unmistakable
immunochemical difference^[^^-^']. Moreover, this applies not
only to the protein synthesis system from prokaryotes but
also to that from e u k a r y o t e ~641.
f~~
Together
?
with the detection
of chimeras of RNA polymerase and of ribosomes, all these
experiments strongly suggest that the entire transcription and
translation systems of prokaryotes are functionally similar
in spite of differences detected between the components from
different bacteria.
Joint functioning of proteins of very different origins has
also been demonstrated in other complex biosynthetic systems.
For instance, the acyl carrier protein in the multienzyme
system of fatty acid synthesis of Escherichia coli can be replaced
by the analogous protein from Agrobacteriurn tumefaciens or
from the chloroplasts of the mesoderm of the avocado pear[65’.
From among the vertebrates, the multicomponent system
required for muscle contraction deserves mention. Here the
Ca2+-bindingtroponine subunit of the rabbit can be replaced
by a corresponding protein from a shrimp[661.
4. Hybrids between Proteins from Prokaryotes and
Eukaryotes
In Nature, a deep rift appears to exist between the prokaryotes, lacking a cell nucleus, and the eukaryotes, which do
have a cell nucleus[671.The question thus arises as to how
Angew. Chem. Int. Ed. Engl.
1 Vol. 15 (1976) N o . 4
5. Conclusions
The numerous examples of chimeras of enzymes which
can be formed in vitro can be supplemented by those arising
in uivo, for example on crossing genetically related yeastsf7’]
or after fusion of somatic cells of such diverse origin as hamster,
mouse, man, or rat[731.Hybrid immunoglobulins made up
of polypeptide chains arising from mouse and man have even
been observed*741.Hence, exchange of subunits between proteins from different organisms is not a special case, but rather
possesses general importance throughout the whole of living
Nature,
How are we to interpret the ability of subunits of widely
differing origin, which vary so much in length, amino acid
composition and sequence, and in their antigenic properties
to mutually replace each other in oligomeric enzymes? Studies
performed on
in particular have shown that
precisely fitting contact surfaces are essential for cooperation
of different polypeptide chains in an aggregate. Accordingly,
it must be suspected that the regions necessary for mutual
interactions and the function necessary for the catalyzed overall reaction are retained by subunits in oligomeric enzymes
across the boundaries of species.
Preliminary evidence was provided by comparative
immunochemical investigations[75! And very strong support
has come from X-ray studies : whale myoglobin and the hemoglobin of an insect larva, which differ in more than 80% of
their amino acid sequence, have almost identical three-dimensional structures[76,771.Some confirmation may also be seen
in the observation that the three-dimensional structure of
185
glycerinaldehyde 3-phosphate dehydrogenase consisting of
four equal subunits is practically identical, whether from a
bacterium or from lobster, although the two enzymes differ
considerably in their amino acid sequences[781.Other functionally related but phylogenetically different proteins likewise
possess very similar structural regionsr791.The three-dimensional structure is apparently more important for function
than amino acid sequence. Hence enzymes can be classified
not only according to function, but also according to their
three-dimensional shape.
The reason for proteins with different amino acid compositions to exhibit very similar folding may be seen in the very
similar influence exerted on three-dimensional structure by
many amino acids. Thus threonine, serine, or valine, leucine,
and isoleucine all have a similar effect on the folding of a
polypeptide chainL8'].They can thus undergo mutual replacement without causing any great change in folding. Evolutionary changes in the amino acid sequence of proteins arising
from different species and yet having the same function are
limited only in so far as that part of the three-dimensional
structure which is essential for functioning is not allowed
to alter. The existence of chimeras of enzymes thus also
becomes understandable, for if the folding of polypeptide
chains has changed so little in the course of evolution then
the subunits of enzymes having the same catalytic activity
will still fit each other spatially, regardless of barriers between
species. The interlocking shape is conserved so well as to
permit association, and, in many cases, catalytically active
cooperation.
Our own studies mentioned in this article were supported
by the Deutsche Forschi~ngsgerneinschaftand the Fonds der
Chemischen Industrie.
Received: September 11, 1975 [A 103 IE]
German version: Angew. Chem. 88. 197 (1976)
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