Note # 3, July 4, 1963

BVaLuaATION QF THE .NTHaReST TO PHYLOGENY OF ESTABLISHING THE allINO.

ACID SkyUANCH OF ACTIVE SITES OF ENZYMES,
— Ewch Fuel fede Ul

We GG. OR P ROTEL MOLROULES tif NAT ARE OF. ILES 2G RL eRe
WIA sO DGRSVEM LIN Lee DO VEO

With respect to problems of phylogeny, studies of sequenceg
around the active gites of enzymes,mey such as performed by Tuppy
in cytochrome co, may be expected to be as a rule the least telling
gtudies, indeed Yene changes in these regdons may be expected to be
very b) smaller than in many other regions of the polypsptide
oie Wh tee Wom
chaind Pioreover what changes are tolerated by mtural gelaction may
be expected to be more limited in kind, The lys preceding the first
and chicken

@ys in horse, bef, pif and galmon/cyt ochrome c is changed to arg
in the silkworm and in yeast, but it appears doubtful from this record
toat an amino-acid other than one of ths wm bagic ones will be found
at this gite in any cytochrome 0. Vasily, the changeg that are
Observed around the active site more probably reflect peculiar
functional requirementa than phylogenetic relationships. Thug, in
cytochromus c, the regidue following the first cys is ala in horse, beef,
Big, galmon and xXtwhweikwm the silkworm, and ger in chicken, Yet, avident-
ly, chicken ig more closely related to horge, pig and beef than is
are the galmon and the gilkworm,

Ag a corollary to (b), it may be stated that whatever
changeg are observed in the wmunmtinwhw functionally most critical region
of the enzyme are most likely one step -substitutiong, since the
chanceg for an intermediary step to be tolerated by natural gelection
are low, In accordance with this congideration the gwitches between

ala and ger and mkm between ala and glu (mentiored in "c) above) invol-

ve one step-mutationg according to the genetic code ag proposed by

JUKaS (1962), The genetic code proposed by SuITH (1962) ig in agreeen
2 note # 3

ment inasmuch ag the alaeser change is concerned, but an ala -
elu change appears here to involve at least two wkxepy mutational
agteps. according to both codes a gereglu changes (or vice-versa)
could be only indirect,

The glu -residue, at this site, is found in yeast only. If
the genetic code in its final vergion should confirm that the
change from glu to ala or to ger cannot bs a one step mutation,
then this fact could be used ag an argument in favor of the
evolutionary independence of the yeast cytochrome c¢ and the
animal cytochwomes c,

Considerations of this kind will make possible statements
about the likelyhood of a phylogenetic relationship between
two functionally analogous proteing belonging to groups thats are
very far removed from gach other. By phylogenetic vetntnin eam p
is meant here the existence of a common ancestor who already per
formed the function of the dea co ndagit molecules. (A wider defini-
tion of phylogenetic kinship igs not ugeful here, since tye possibi~
lity cannot be excluded that all proteing, animal and vegetal, have
a common ancestor),

Functionally analogous proteging belonging to vastiy different
forms of life may share a few characters of sequence and differ in
all other characters of sequence. The qwastion then arises whether
the similarities have arisen by convergence from evolutionarily
unrelated polypeptides, or whether a common poly pe ptide amcestor
enuowed with a similar function has existed, but the gimilaritiegs
in seqwnce have wpymwatpe@ mogtly been wiped out by divergent
evolutionary change. If changes are observed at the active sites
of snzymueg that cannot be due to one-step mutations, then the first
hypothesis can be stated to be the more likely, Its likelyhood will
be the greater the more guch differences are found between the two

forms of the enzymethat are compared. The study of the amino-~
3 Note # 3

acid sequence in closely or moderately related forma is, ag gtated,

of little interest to phylogeny, but on the other ham, if forma are
examined that are extremely far removed from each other on the evo-

lutionary scale, theie critical regiong of the molecule are probably
the most informative.

One may put it this way : If a common ancestry of two
polypeptide chains is already ascertained by a sufficient nupber of
structural homologies, the indirectness of an amino-acid subg tit ution
Will indicate a degreg of functional tolerance to change at a given
molecular gite; if the common ancestry ig not egtablished, then the
indirectnegg of ang amino-acid substitution at a molecular gite
presumed to be very intolerant to change representa some evidence
in favor of the contention that the two polypeptide chaing do not

have a vommon ancestor (in the sense defined above),

Regarding extra-terrestrial life : If &ife on Marg
should prove very gimilar to that on earth; if it is based on the
game genetic code and on the activity of proteinaceaous enzymes
that carry out the same fundamental functions; if, furthermore, in
& great number of these enzymes, whose function has been ascertained
and found identical with that of corresponding telluric enzymes,
amino-acid substitutiong are noted at molefular giteg that are foum
invariable on earth; if, finally, these gubgtitutions, according to
the genetic code, cannot have been one step mutations; then we ghall
have grounds for believing that living matter on earth and on Mara,
althowh very similar, has not had a common origin, but must have
arisen independently.

Thesé@ consicerations possibly furnish the best criterion to

date for making a tentative statenenfabout the independent origin

of nearly identical forms of life,
July 4th, 63
Note # 2

q@UALIFICATION OF NOTH 1 OF JULY grd

AN agsociation of chemical units that is completely random must
not be expected to be found in wacromolecules of either chemogenic

or biogenic origin ;

1) Macromolecules of chemogenic origin,

a) The proportions of the different chemical units that make
up such macromolecules cannot be expected to be equal in the original
reaction mixture, hence the chemogenic prodwt will be non-random in
the seme of the predominance of some units over others.

b) The condensation of the chemical units that make up the
macromolecule should perhaps not be expected to lead ugually to
products whose composition is very close to that of the original
reaction mixture (cf. experimental reaults of Fox and Harada, 1960),

ec) (Corollary to (b)): Certain aggociationg between units, Hex
i.e. certain neighbours in the macromolecule, may not occur, or occur to

a gmall extent, for chemical reasons,

2) Macromolecules of biogenic origin.

a) Cn earth, the proportiong of the different amino-acidg in pro-
teins, congiderim, average figurea of a number of them in different
groups, are not random (cf. Vegotsky and Fox, 1962).

b) Mutations, except for the probable inherent ingtability of
some of them, probably tem to introduce complete randomness, but
this trem is counteracted by natural selection,

c) (= (c) of paragraph (1) ). Even under conditions of enzymatic
gynthegig certain gequences may be excluded for chemical reasons,
guch ag perhaps the wayxmmea association of glu and pro (cf. Vegotaky

and Fox).
2 Note # 2

The question arises whether, between certain limits,
a more highly randomized seqiwnce, given a certain number of
distinct bagis ahemical units, may not be more indicative of the
biogenic origin of a macromolecule than a legg randomized geqiwne
(be cause of the trend toward greater randomization through mutation

in "primary" macromolecules, as compared to chemogenic condengation),