are two subjects that I am deeply
Meted in—structure, the detailed
fe of molecules, crystals, and cells, de-
din terms of their constituent atoms,
Pinteratomic distances determined to
00.01 A., an interest that began in
pith and has received most of my at-
pm until recent years; and the basis
physiological activity of substances,
erest that is more recent but just as
Itis with a deep feeling of satisfac-
Maat I have reached the firm conclu-
fo recent years that these two fields
must intimately related.
By have we still so little understand-
the structural basis of the physio-
activity of chemical substances, des-
me interest and effort of many able
Pogists and chemists during recent
s} I believe that it is because the
fm has been examined, in the main,
She point of view only—not the
Pe Point of view, but one which, un-
Gives a vista insufficient to reveal
FP complex nature. This point of
® that which surveys the chemical
' ty of molecules—their tendency to
their chemical bonds, the very strong
tween atoms, and to form new
™ bonds. The other point of view
SS teeded is that which directs the
 fYe to the detailed size and shape
. Nolecules and the nature of the
ti actions of molecules with other
‘12 particular with the macro-

     
 
 
   
 
  
  
 
 
   
 
   
 
  
  
 
 
    
 
 
   
    
 
 
  
   
   
 
   
 
   
 

mo which characterize the living
Until very recently physiolo-
Pharmacologists have barely
a ‘o this aspect of their great prob-
: &m convinced that once they
this new idea seriously a
“€ greatest development. will
a, J believe that the next
; medi be as great years for biol-
‘pp. ine as the past twenty have
Ysies and chemistry.

* ok ok & & & & Biological Science ~ % & kk kK *

Molecular Architecture and
Biological Reactions

Linus PauLine

Chairman, Division of Chemistry and Chemical Engineering,
California Institute of Technology, Pasadena, Calif.

Answers to many basic problems of biology—nature of growth,
mechanism of duplication of viruses and genes, action of enzymes,
mechanism of physiological activity of drugs, hormones, and vita-
mins, structure and action of nerve and brain tissue—may lie
in knowledge of molecular structure and intermolecular reactions

Kwek aK KK KK KK Kh KK KK KKK

Eddington has said that the study of the
physical world is a search for structure
rather than a search for substance. If we
ignore the philosophical implications of the
words, we may say that the chemist and
biologist in their study of living organisms
must carry on both a search for structure
and a search for substance, and that the
second of these must precede the first.
Investigators have had great success in
isolating chemical substances from living
organisms, and in determining the chemi-
cal composition of the simpler of these sub-

stances. The chemical composition is also’

known of many substances of external
origin which exert physiological activity
on living organisms. We may consider
this work of isolation and identification of
active chemical substances as the search
for substance in biology.

The Search for Structure

The search for structure has also made
great progress. From the one side biolo-
gists have, by visual observation with the
microscope, made thorough studies of the
apparent structure of aggregates of cells, of
cells themselves, and of certain constitu-
ents of cells, such as chromusomes. This
visual observation has provided informa-
tion about structures in size extending
down to 1074 cm., 10,000 A Forty years
ago the dark forest of the dimensional un-
known stretched from this limit of the
visible microscope back indefinitely into
the region of smaller dimensions. In re-
cent years the region from 1077 down to
10-# cm., containing atoms and simple
molecules, has been thoroughly explored by
an expedition outfitted with x-rays and
similar tools, and the physicists are
strongly pushing back into the region of
the structure of atomic nuelei, below 107 !?
em. Another detailed exploration is being
carried out with the electron microscope.
This has pushed the nearer boundary of
the unknown back from 1074 to 107 em.,

a
"ME 94 NO. 10+ ++ » MAY 95, 1946

although the major portion of this region
has been only sketchily explored during the
few years since the development of the
electron microscope, and a very great
amount of work still remains to be done.

The answers to many of the basic prob-
lems of biology—the nature of the process
of growth, the mechanism of duplication
of viruses, genes, and cells, the basis for
the highly specific interactions of these
structural constituents, the mode of action
of enzymes, the mechanism of physiologi-
cal activity of drugs, hormones, vitamins,
and other chemical substances, the struc-
ture and action of nerve and brain tissue—
the answers to all these problems are
hiding in the remaining unknown region
of the dimensional forest, mostly in the
strip between 10 and 100 A, 10-7 and
10-*cm.; anditis only by penetrating into
this region that we can track them down.

There are many ways of investigating
this region—by x-rays, ultracentrifuges,
light-scattering techniques, the study of
chemical equilibria, the techniques of
degradation, isolation, identification, and
synthesis used by the organic chemist,
serological methods, chemical genetics, the
use of both radioactive and nonradioactive
tracers, the use of electron microscopes of
improved resolving power-——but no one
method is good enough to solve the prob-
lem, and all these methods must be applied
as effectively as possible if the problem is
to be solved.

At the present time we know in com-
plete detail the atomic structure of many
simple molecules, including a few amino
acids; but we do not know in detail how
the amino acids are combined to form pro-
teins. We do not know, except very
roughly, even the shapes of such important
molecules as serum proteins, enzymes,
genes, the substances which make up
protoplasm—and if we are to obtain a
thorough understanding of the structure of
living organisms detailed information

1375
apout the atomic arrangement of these
substances must be obtained.

Let us imagine ourselves increased in
size by the linear factor 250,000,000—the
commonly used factor in molecular models,
which makes 1 A, 10-8 cm., become ap-
proximately 1 inch, atoms on this scale
being 2 or 3 inches in diameter. With this
magnification we would become about
equal in height to the distance from the
earth to the moon. Let us consider our-
selves examining the earth, which would
appear to us to be about the size of a bil-
liard ball; and let us concentrate our at-
tention on a small organism on the surface
of the earth—-New York City—which
would appear as a spot about 0.01 inch in
diameter, barely visible to the naked eye,
and showing itself to be living by slow
changes in shape and size.

To obtain a better view of this organism
we could use a microscope, the resolving
power of which would be about 1,000 feet;
we could distinguish Central Park, the

rivers, and such aggregates of skyscrapers -

as Rockefeller Center, but the individual
skyscrapers would not be clearly defined.
By “chemical” methods we would know
that, running through the veins and arter-
ies of this organism, there were substances
such as street cars, busses, automobiles,
ships, and people; and we might, by the
use of membranes of known pore size Or
by some similar method, obtain the molec-
ular weight of these. In addition, we
would have obtained, through the applica-
tion of a strange method of experimental
investigation, the diffraction of x-rays and
electron waves, complete information
about the structure of objects smaller than
about 1 foot in diameter, such as a storage
battery, a small electric motor, @ piece of
cable, a small gear wheel, a bolt or rivet.

The use of the electron microscope, with
resolving power about 10 feet, would give
us very much additional information. We
would know exactly—that is, to within 10
feet—the shape of the Empire State Build-
ing, though we might not be sure about
the separate smaller rooms into which it
is divided, and we could not obtain by the
electron microscope information about the
elevators and the machinery for operating
them, the steel girders of which the build-
ing is constructed, and other structural
features of similar size. We would be able
to see, with the electron microscope, an
automobile only as a particle, barely dis-
cernible, and roughly spherical in shape,
and the human beings in the city would
not be visible. We could get complete in-
formation about a storage battery, & ring
gear, a brake pedal—but not about the
automobile built up of these and many
other parts; and it is clear that to obtain
an understanding of the structure of this
city we would still need to find a method of
exploring objects in the range 1 to 10 feet.

Our hope for achieving precise knowl-
edge about biological structures and reac-
tions is based largely on the electron micro-
scope and on diffraction methods. The dif-

1376

fraction studies of simple molecules have
been carried out in sufficient number to
permit the formulation of generalizations
about atomic radii, bond angles, and other
features of molecular configuration; it is
still very important that the exact struc-
ture be determined of vitamins, bacterio-
static agents, and other physiologically
active substances—the complete crystal
structure determination of the rubidium
salt of penicillin so ably made by Dorothy
Crowfoot and Barbara Rogers-Low (6) has
provided not only decisive information
about the chemical formula of the sub-
stance but also the structural basis for
later consideration of the detailed mecha-
nism of its bacteriostatic activity.

Structure of Protein

The most important of all structural
problems is the problem of the structure of
proteins: until this problem is solved all
discussions of the exact molecular basis
of biological reactions remain in some de-
gree speculative. The polypeptide-chain
structure of proteins proposed by Fischer
is now generally accepted, and there is
little doubt that the picture of folded
chains held by hydrogen bonds, van der
Waals forces, and related weak interac-
tions in more or less well-defined configura-
tions, as discussed eleven years ago by
Mirsky and me (9) is essentially correct.
But this whole picture remains very
vague—for only a few proteins (such as
g-lactoglobulin (3)) do we have nearly

_complete knowledge of the numbers of

residues of the different amino acids in the
molecule, and for no protein does there
exist more than fragmentary information
either about the sequence of the different
residues in the polypeptide chain or about
the way in which the chain is folded. Only
for fibrous proteins in the completely ex-
tended state do we have knowledge (still
very rough) of the configuration and rela-
tive orientation of the polypeptide chains
(as originally determined by Astbury), and
this knowledge applies only to the back-
bone of the chains and not to the side
groups. There is urgent need for complete
and accurate structure determinations of
proteins and related substances. So far
these determinations have been’ reported
for only four such substances (5)—two
amino acids and two simple polypeptides—
all made in our Pasadena laboratories; and
it is my hope that, now that the war is
over, precise information will rapidly
accrue, including ultimately detailed struc-
tures of fibrous proteins, respiratoly pig-
ments, antibodies, enzymes, reticular
proteins of protoplasm, and others.

Importance of Shape

Despite the lack of detailed knowledge of
the structure of proteins, there is now very
strong evidence that the specificity of the
physiological activity of substances is de-

CHEMICAL AND ENGINEERING NE

termined by the siz

cules, rather than. ona shape e
hemical ; Marly ge;
chemical properties, and thar §
shape find expression by q the sig pot
extent to which certain ute miting &
two molecules (at | ACE Tegiags

, east: one me

usually a protein) can be ne i
juxtaposition—that is, the oxtee ine,
these regions of the two mmo .
complementary in structure 0)

nation of specificity in terms of &
key” complementariness jg q oe
Ehrlich, who expressed it often, ta
such as “only such substanes
anchored at a particular part of th be
ism which fit into the molecule of the Organ.
ent combination as 4 piece of m € recip
into a certain pattern’. SALE fity

In recent years the concept of com
mentariness of surface structure of ang:
and antibody was emphasized B
and Haurowitz (4), Mudd (10) ad i]
ander (1), and then was strongly sup _
by me (11) in the course of an effort
understand and interpret serological re
nomena in terms of molecular structuny
and molecular interactions. Since 1%
my collaborators (Dan H. Can
David Pressman, Carol Ikede, L g
Pence, G. G. Wright, 8. M. Swingle, Dg
Brown, J. H. Bryden, A. L. Gromsty
L. A. R. Hall, Miyoshi Tkawa, Prog
Lanni, J. T. Maynard, and A. B. Pardea)
and I have gathered a great amount qf
experimental evidence about antigg,
antibody interaction (12), which not only
supports the general thesis that serologisl
specificity is the consequence of structural
complementariness, but provides informe.
tion about extent of complementarinems,

It has been verified that the closenseg
of fit of an antibody molecule to its homal
ogous haptenic group is to within better
than 1 A.—that a methyl group (van de
Waals radius 2.0 A) can replace a chlorime
atom (radius 1.8 A) in a haptenic grow
with little interference with its combim
tion with antibody (as was first shown by
Landsteiner), but that interference i
caused by replacing 4 hydrogen stom
(radius 1.2 A.) by a methyl group. Th
complementariness in structure with t
spect to proton-donating and prot
accepting hydrogen bond-forming grou”
has been found to be very important &
determining the strength of attraction €
antibody and haptenic group; and te
complementary electrical charge in ane
body homologous to the p-acophenyti
methylammonium group has been show,
to be within about 2 A. of the minima,
possible distance from the charge of 0 gj
site sign in the haptenic group. The gars
amount of quantitative data which bai,
been gathered for scores of different Sg.
tens and antigens and successfully 150"
preted in terms of molecular structure ai ‘
the concept of complementariness | er
no doubt that this structural expo"
of gerological specificity is correct. :

The phenomenon of specificity, 9° *
mon in biology, is rare in chemistry

in w

  
  
  
    
   
   
   
 

a
ole general exception mentioned be-
*: Only very occasionally does there
ff. unique representative of a class of
mounds, such as the ion W,Cl,-—-,
hn owes its special stability to the
, of radii of the atoms of chlorine and
sitive tungsten, which permits a co-
4 bond to be formed between the two
4en atoms in the complex. The one
al chemical phenomenon with high
Bcity is closely analogous in both its
re and its structural basis to biological
ficity: this phenomenon is crystal-
fon. There can be grown from a solu-
eeontaining molecules of hundreds of
ent species, crystals of one substance
bare essentially pure. The reason
e great specificity of the phenomenon
Bystallization is that a crystal from
mone molecule has been removed is
closely complementary in structure to
molecule, and molecules of other kinds
ot in general fit into the cavity in the
al or are attracted to the cavity less
gly than a molecule of the substance
Only if the foreign molecule is
similar in size and shape and the
fon and nature of active (hydrogen
ki-~forming) groups to the molecule it is
Bcing will it fit into the crystal; and
Bindeed found that the tendency to
Peolution formation depends upon the
estructural features (such as replace-
tof a chlorine atom by a methyl group)
he tendency to serological cross reac-

   
  
   
 
 
 
  
   
     
 
 
  
 
   
   
 
 
  
 
 
    
   
   
   
  
   
  
   
    
  
  
 
  
  
  
   
   
 
  
   
    
  

inples of Biological Specificity

pay isolated examples of biological
Micity and biological similarity deter-
ped by molecular size and shape and the
pita nature of intermolecular forces
Pt be mentioned, such as the similarity
Physiological (antipyretic-antineural-
activity of 4-isopropylantipyrine and
Muethylaminoantipyrene (pyramidon),
Ru is clearly the result of the similarity
rf and shape of the isopropyl group
the dimethylamino group. I shall,
‘ver, discuss in detail only the speci-
PY of enzymatic reactions.

fom the standpoint of molecular
€ and the quantum mechanical
of chemical reaction, the only rea-
ule picture of the catalytic activity of
es is that which involves an active
V of the surface of the enzyme which
, Rely complementary in structure not
* substrate molecule itself, in its
al Configuration, but rather to the
Pale molecule in a strained configura-
“Corresponding to the “activated
*x” for the reaction catalyzed by
“4yme: the substrate molecule is
Son to the enzyme, and caused by
. es of attraction to assume the
, State which favors the chemical
that is, the activation energy of
“ction is decreased by tle enzyme to
F extent as to eause the reaction to

proceed at an appreciably greater rate
than it would in the absence of the enzyme.
This is, I believe, the picture of enzyme
activity which is usually accepted.

Experimental data have not been gath-
ered which permit the induction of so pre-
cise a representation of the structure and
configuration of the active region of any
enzyme as for the antibodies discussed
above, but there do exist some data which
support the general concept. If the en-
zyme were completely complementary in
structure to the substrate, then no other
molecule would be expected to compete
successfully with the substrate in combin-
ing with the enzyme, which in this respect
would be similar in behavior to antibodies;
but an enzyme complementary to a
strained substrate molecule would attract
more strongly to itself a molecule resem-
bling the strained substrate molecule than
it would the substrate molecule. Ex-
amples of this behavior have been found:
the hydrolysis of benzoyl-l-tyrosylglycine
amide by either chymotrypsin or papain
was found by Bergmann and Fruton (2) to
be practically completely inhibited by an
equal amount of benzoy]l-d-tyrosylglycine
amide. This suggests that the strained
configuration of the /-isomer during the
enzymatic hydrolysis is somewhat similar
to the normal configuration of the d-isomer.

More extensive quantitative studies of
inhibition of enzyme activity might well
provide very interesting information about
the configuration of the enzyme molecules.
Carl Niemann and I have studies of this
kind under way.

It is highly probable that many chemo-
therapeutic agents exercise their activity
by acting as inhibitors to an enzymatic re-
action through competition with an essen-
tial metabolite of similar structure. It
was shown by Woods (16) in 1940 that the
bacteriostatic action of sulfanilamide re-
sults from an inhibitory competition with
p-aminobenzoic acid, and can be overcome
by increasing the concentration of the lat-
ter substance. The metabolite and its in-
hibitor are closely related in molecular
shape, differing in the replacement of a
carboxyl group by a sulfonamide group.
Other pairs in which a carboxy! group is
replaced by a sulfonic acid or sulfonamide
group are nicotinic acid and pyridine-3-
sulfonic acid or its amide (7), pantothenic
acid and pantoyltaurine (14) and the a-
aminocarboxylic acids and the correspond-
ing a-aminosulfonic acids (8).

An interesting case of inhibition is that
of thiamine by pyrithiamine (13), ‘the
corresponding substance with the 6-mem-~
bered pyridine ring in place of the 5-mem-
bered thiazole ring. The effective competi-
tion of pyrithiamine with thiamine for
combination with the enzyme or other
macromolecule involved might well have
been predicted from the known cross
reactivity of aromatic 5-membered rings
containing sulfur and 6-membered rings
not containing sulfur, as is strikingly
shown by the formation of solid solutions

UME 94, NO. 10+ +» » MAY 25, 1946

by thiophene and benzene. An analogous
Situation has been reported (16) by D. 8.
Tarbell of the University of Roehester.
He has found that any substitution in the
benzenoid ring of 2-methylnaphthoquinone
destroys its vitamin K activity, but that
the substance with a sulfur atom in place
of —CH=—=CH— in the benzenoid ring
retains this activity. These facts indicate
that in the process of exerting vitamin K
activity the benzenoid end of the molecule
must fit into a pocket carefully tailored to
it; that the other end is not so surrounded
is shown by the retention of activity on
changing the alkyl group in the 2-position.
On the other hand, the failure of pyrithi-
amine to replace thiamine as a metabolite
indicates that the sulfur atom of the
thiazole ring in thiamine not only is ef-
fective in binding the molecule into its seat
of action but also takes part in some way
in the subsequent chemical reactions in-
volved in the metabolic process.

Many Sciences Cooperate

The complete understandiag of physio-
logical activity will require consideration
not only of molecular structure and weak
intermolecular forces, but also of the
chemical reactivity of the substances and
of such other properties as solubility in
different phases and degree of ionization,
as well as of those properties of living
organisms which may long defy simplifica-
tion to chemical description; the impor-
tance of the problem for practical medicine
as well as for fundamental biology is so
great as to justify the attention and effort
of many workers, in various fields of sci-
ence, through whose cooperative effort the
solution will some day be found.

Literature Cited

(1) Alexander, J., Protoplasma, 14, 296
(1931).

(2) Bergmann, M., and Fruton, J. &.,
J. Biol. Chem., 138, 124, 321 (1941).

(3) Brand, E., Saidell, L. J., Goldwater,
W.H., Kassell, B.. and Ryan, F. H.,

: J. Am. Chem. Soc., 67, 1524 (1945).

(4) Breinl, F., and Haurowitz, F., 2.
physiol. Chem., 192, 45 (1930).

(5) Corey, R.B., Chem. Rev., 26, 227 (1940).

(6) Crowfoot, D., and Rogers-Low B.,
mentioned in Science, 102, 627 (1946).

(7) Mellwain, H., Brit. J. Exptl. Path., 21,
186 (1940).

(8) Mcllwain, H., J. Chem. Soc., 1941, 75;
Brit. J. Exptl. Path. 22, 148 (1941).
Mirsky, A. E., and Pauling, L., Proc.

Nat. Acad, Sci., 22, 439 (1936).

(10) Mudd, Stuart, J. Immunol., 23, 423
(1932).

(11) Pauling, L., J. Am. Chem. Soce., 62, 2643
(1940).

(12) Pauling, L., and collaborators, J. Am.
Chem. Sec., 68, 250 (1946), and earlier
papers.

(13) Robbins, W. J., Proc. Nat. Acad. Ser.,
27, 419 (1941); Woolley, D. W., and
White, A. G. C., J. Exptl. Med., 78,
489 (1943).

(14) Snell, E. E., J. Biol. Chem., 139, 975,
141, 121 (1941).

(15) Tarbell, D. S., private communication
to author.

(16) Woods, D. D., Brit. J. Exptl. Path., 21,
74 (1940).

(9

1377