COMMENTARY
INBORN
ERRORS
OF
METABOLISM:
SOME
THOUGHTS
ABOUT
THEIR
BASIC
MECHANISMS
901
Piiiu tries
VOLUME 45 JUNE 1970 NUMBER 6
T
HE question of whether, in the inbornerrors of metabolism, genetic
informa-tion is absent or coded incorrectly
(struc-tural mutations), or whether the gene itself
is normal l)ut is not activated (regulatory
defects) is extremely important in the
di-rections taken for the future consideration
of such problems. The problem of
introduc-ing new, ve1l defined, genetic information
into a human cell which is either lacking
such information or has abnormal genetic
information appears formidable at the
cur-rent time. However, the existence of
regula-tory defects would imply that the correct
information to make the deficient en-zyme (s) is available to the cell, and we
need only find ways of activating
(induc-ing) this genetic information. Enzyme
in-duction has been clearly demonstrated to
occur in the intact animal, and there is a
growing body of information about its
mechanism (s).
Recently, investigators have demonstrated
reduced, but not absent, enzyme activity in
tissues obtained from certain patients with
inborn errors of metabolism. Exhaustive
study of the residual enzyme activity,
which might establish either a reduced
quantity of a normal enzyme or a
structur-ally abnormal enzyme which functioned
be-low normal, has usually not been made.
Also, from the many laboratories now
studying tissues from patients suspected of
having genetic metabolic disease, there are
reports of complex situations in which more
than a single enzyme activity is found to be
deficient.1 The easiest explanation for these
diseases with reduced tissue enzyme
activ-ity or multiple enzyme deficiencies has
been to consider them “regulatory” defects.
Although my comments are directed at this
interpretation of the inborn errors of
me-tabolism in light of our current information
as to how protein (enzyme) synthesis
might be regulated in man, they begin by
acknowledging our debt to bacterial genet-ics.
Because one can perform very large
num-bers of experiments over many generations,
virtually all the pioneering studies in
ge-netic regulation have been carried out in microorganisms. The extraordinary
ad-vances in the understanding of the genetic
regulatory mechanisms have depended on
the use of bacteria. The magnitude and
ra-pidity of enzyme induction and repression
in bacteria are usually much more
impres-sive than in animal tissues. Moreover, the
experimenter can select bacteria with
muta-tions affecting only regulatory functions.
Theories derived exclusively from the
study of regulatory mechanisms in bacteria
have led to the proposal of mechanisms for
many single and multiple enzyme
defi-ciencies in man. It is perfectly appropri-ate and indeed exciting to think in this framework as long as we keep clearly
be-fore us the fact that we have little
tion in higher species to suggest that the
mechanisms for induction and repression of
gene products are the same in man as in
bacteria.
In some instances, but not always, genes
which control related functions (e.g., the
synthesis of a group of enzymes controlling
a metabolic pathway ) in bacteria are
grouped together on the chromosome and
operate as a unit, called, therefore, an
op-2 J has been readily assumed by some
that operons exist in man, and indeed they
may. However, the operon is a genetic
def-inition. In order to say that the synthesis of
a group of enzymes is governed by an
op-eron, we must have an accurate genetic
map showing that the areas controlling
these enzymes are adjacent on a
chromo-some; and, that this group of genes does
in-deed function as a unit. The fine structure
genetic analysis required to prove this
can-not now be done in man.
To return to what is known of that species,
certain disorders of man in which enzyme
activity is reduced (but not absent) have
first been considered regulatory gene
de-fects and later shown to be structural gene
mutations. This is well illustrated by the
studies of a rare genetic defect, the Swiss
form of acatalasia, which was originally
considered a regulatory defect and is now
considered a structural defect by Aebi.3 In
this condition, activity of the enzyme
cata-lase, a widely distributed enzyme in human
tissues, is reduced in erythrocytes to about
0.1 to 1.3% of normal. Aebi3 originally
found that the small amount of residual
cat-alase activity isolated from deficient
pa-tients behaved similarly to normal catalase
during column chromatography (Sephadex
G-100), during electrophoresis on starch
gel, and by precipitation with anticatalase
antibody in an Ouchterlony double
diffusion test. After these studies, they quite
properly suggested the possibility that this
disorder might represent a regulatory gene
defect. Such an interpretation would
sug-gest that the information to make normal
catalase was present in the cell and that
therapy, if desirable, should center around
efforts to induce new enzyme formation.
Realizing these implications, Aebi3
contin-ued his efforts to characterize the residual
catalase activity more thoroughly. Using a
new column chromatographic technique
(calcium phosphate-DEAE cellulose) , he
again isolated catalase from mutant and
normal cells. In the new preparations, in
which catalase is less likely to be
dena-tured, there was a distinct difference in
electrophoretic mobility as well as in heat
stability between the mutant enzyme and
normal catalase. These findings suggest that
the mutant catalase is, after all, structurally
abnormal; they rule strongly against a
regu-latory gene defect. To demonstrate these
differences was a great, but important,
ef-fort.
When exhaustive study has been made of
the residual enzyme activity in certain other
genetic enzyme deficiencies in man,
abnor-mal proteins have likewise been found;
such findings strongly suggest structural
gene mutations. The recent work of
Ted-esco and Mellman4 has shown that the
en-zyme arginosuccinate synthetase in
fibro-blasts derived from a patient with
citrul-linuria has much less affinity for its substrate
(citrulline) than does the normal enzyme.
This clearly supports a structural mutation
in this enzyme protein. It is also interesting
that the enzyme, although inactive when
as-sayed in vitro under normal circumstances,
functions normally at high citrulline
con-centrations. Without these exhaustive
stud-ies, it might have been concluded that
ar-ginosuccinate synthetase was absent
be-cause of defective activation of the gene
responsible for its synthesis. As discussed
by Scriver,5 substrate accumulation could
be thought of as representing an adaptation
by the host to the mutant enzyme and may
explain the ability to synthesize urea in
these situations.
Dual enzyme defects have been
demon-strated or suspected in some diseases of
which the rare condition, congenital orotic
aciduria, has perhaps been most carefully
studied. These dual enzyme defects have
af-COMMENTARY
feeling an operon. Tissues from patients
with orotic aciduria, and fibroblasts
devel-oped from them and grown in vitro in a
controlled environment, are deficient in two
sequential enzymes required for de novo
pyrimidine biosynthesis.6 Pinsky and
Krooth7 have shown that certain drugs in
vitro will markedly increase the activities of
both these deficient enzymes. This can be
interpreted as gene activation. The fact that
two sequential enzymes are deficient while
both enzyme activities can be enhanced in
vitro has suggested the operator defect
hy-pothesis mentioned here. The inability to
perform fine structure genetics currently
precludes confirmation or denial of that
hy-pothesis. The widespread impression that
the dual enzyme deficiency of orotic
acidu-na represents a regulatory defect would be
strengthened enormously if the small
amount of residual enzyme activity in these
cells was shown after exhaustive study to
be identical to those enzymes present in
normal cell lines.
Another entirely different type of
meta-bolic regulation has recently received
con-siderable attention in the discussion of
genetic diseases in man. This type of
regula-tion occurs not at the nuclear level
consid-ered in this Commentary but by affecting
the activity of enzymes already present.
Wyngaardens has suggested that
overpro-duction of uric acid in certain patients with
gout results from abnormalities in the
regu-lation of the activity of the first specific
en-zyme in uric acid biosynthesis. The recent
work of Rosenbloom, et al. suggests that the
enzyme defect in the Lesch-Nyhan
syn-drome provides excess substrate
(5-phos-phoribosyl-1-pvrophosphate) for de novo
uric acid biosynthesis and hence
overpro-duction of uric acid. In this situation, the
in-creased availability of a substrate enhances
the rate of uric acid synthesis. These
exam-ples of ctoplasmic regulation do not involve
the synthesis of new enzyme molecules.
Early in this Commentary it was pointed
out that theories of genetic regulation as
studied in bacteria must be applied with
caution to man. A comparison of certain
properties of bacterial and of liver cells
in-dicates that regulatory mechanisms in the
latter species may indeed differ from those
observed in #{176}Growing
bacteria may divide about every half hour,
whereas the normal liver cell of man has a
lifespan which may be of the order of
months to a year. In bacteria, enzymes
(
proteins)
are not degraded oncesynthe-sized; though, after the induction of
addi-tional enzymes, the induced enzymes return
to their basal levels by exponential dilution
with new bacteria which contain very little
of the enzyme.’#{176} On the other hand, in the
liver, following enzyme induction, enzyme
(protein) molecules are exported or
de-graded within the cell in order to return the
enzymes to their basal activity values.
Cer-tain liver enzymes (e.g.,
tyrosine-r-ketoglu-tarate transaminase) are renewed and
degraded very rapidly with a half-life of
several hours.1#{176}Ribosomal RNA, a vital
com-ponent of the cell’s machinery to synthesize
proteins, is renewed rapidly with respect to
cell life in mammalian (rat) liver.hl
How-ever, in dividing bacteria ribosomal RNA
turns over slowly, if at all, with respect to
the generation time of the cell.
The rate of synthesis of a given enzyme
in bacteria is thought to be determined only by the concentration of its specific
messen-ger RNA (mRNA) in which is coded the
information for the synthesis of specific
en-zymes.22 Bacterial mRNA has a very short
life span, of the order of several minutes.’3
By contrast, in the liver most of the mRNA
is stable for much longer times.’4”
Actu-ally, there may be more than one class of
mRNA in mammalian cells. Studies of
hor-monal regulation (by hydrocortisone) of
the synthesis of tryptophan pyrrolase and
tyrosine--ketoglutarate transaminase in
liver have suggested that the concentration
of mRNA alone does not always determine
the rate of enzyme synthesis.16 In this
in-stance, regulation of the synthesis of these
enzymes, at least in part, may occur beyond
the level of gene transcription. It is
sug-gested that factors may regulate not only
INBORN ERRORS
from DNA
(
gene transcription)
butsubse-quently the rate at which the mRNA codes
for protein. Studies in mammalian cells
de-rived from liver tumors and growing in
vi-t,o have similarly suggested that regulation
of enzyme synthesis may occur beyond the
level of gene transcription.’7 These studies
suggest that, in mammalian tissues, unlike
the situation in bacteria, important controls
on the rate of enzyme synthesis may be
cx-erted beyond the stage of mRNA synthesis.
Another mechanism by which the
quan-tity of an enzyme in mammals can be
in-creased is by reducing the rate at which the
enzyme is degraded while maintaining a
constant rate of its synthesis. The
degrada-tion of the enzyme tryptophan pyrrolase
(TP) in mammalian liver is reduced in the
presence of high concentrations of its
sub-strate, the amino acid tryptophan. By
in-jecting large quantities of this amino acid
into rats, the amount of TP in their livers
can be greatly increased due to the reduced
rate of TP degradation in the face of a
usual rate of synthesis.15
From these considerations, it is suggested
that metabolic controls in higher organisms
may be of a different nature from
regula-tion of bacterial metabolism. Although
ani-mal cells are not exposed to drastic changes
in environment, their activities must be
in-tegrated with a variety of very
heteroge-nous cells. Therefore, regulatory
mecha-nisms in mammals might be much more
complex than in bacteria.’ Yet, the
differ-ences themselves may be instructive.
The study of control mechanisms in man
must, then, be based on diploid mammalian
cells, though they may rely heavily on
mod-els from simpler organisms, which are so
ex-tremely well suited for laboratory
investiga-tion.
The ability of experimenters to form
hy-brids between human cells in vitro might
prove to be a useful tool in elucidating the
question of mammalian regulatory defects2o
For instance, if a cell is deficient in an
enzyme activity, and after hybridization in
vitro with another cell the original mutant
cell begins making enzyme (identifiable by
different properties from that of its
partner
)
, one could assume that somethingpresent in the cell to which the mutant cell
was mated has turned on
(
activated)
apreviously inactive gene in the original mu-tant cell.
Although not rigidly proved, there are
cx-amples in man where enzyme induction
might well have occurred. The studies of
Yaffe and coworkers2l and Crigler and
Gold2’ strongly suggest that the reduction
of serum bilirubin concentrations in their
pa-tients with congenital nonhemolytic
uncon-jugated hyperbilirubinemia (Crigler-Najjar
Syndrome), is the result of enzyme
induc-tion (glucuronyl transferase) by
phenobar-bital. If indeed regulatory defects exist in
man, enzyme induction might well be the
first rather specific use of genetic
engineer-ing in man.
Pioneering explorations by means of
bac-terial genetics have opened the territory
now under examination, and properly
eval-uated studies with microorganisms will
con-tinue to have great usefulness. However,
the basic mechanism of inborn errors of
hu-man metabolism will need to be revealed in
mammalian cells. Some aspects of that
process and its possible implications have
been presented in the foregoing brief
re-view.
R. RODNEY HOWELL, M.D.
Department of Pediatrics
Johns Hopkins University
School of Medicine
Baltimore, Maryland 21205
REFERENCES
1. Auerbach, V. H., and DiCeorge, A. M.:
Ge-netic mechanisms producing multiple
en-zyme defects. A review of unexplained cases and a new hypothesis. Amer. J. Med. Sci., 249:718, 1965.
2. Jacob, F., and Monod, J.: Genetic regulatory mechanisms in the synthesis of proteins, J. Molec. Biol., 3:318, 1961.
3. Aebi, H.: The investigation of inherited
en-zyme deficiencies with special reference to
acatalasia. In Crow, J. F., and Neel, J. V.,
ed.: Proceedings of the Third International
Congress of Human Genetics. Baltimore: Johns Hopkins Press, p. 189, 1967.
Argino-succinate synthetase activity and citrulline metabolism in cells cultured from a citrulli-nemic subject. Proc. Nat. Acad. Sci. U.S.A., 57:829, 1967.
5. Scriver, C. R.: Inborn errors of amino-acid
me-tabolism. Brit. Med. Bull., 25:35, 1969. 6. Howell, R. R., Klinenberg, J. R., and Krooth,
R. S.: Enzyme studies on diploid cell strains
developed from patients with hereditary oro-ticaciduria. Johns Hops. Med. J., 120:81, 1967.
7. Pinsky, L., and Krooth, R. S.: Studies on the control of pyrimidine biosynthesis in human
diploid cell strains. I. Effect of 6-azauridine
on cellular phenotype. Proc. Nat. Acad. Sci. U.S.A., 57:925, 1967.
8. Wyngaarden, J. B.: Gout. In Stanbury, J. B.,
Wyngaarden, J. B., and Fredrickson, D. S.,
ed.: The Metabolic Basis of Inherited Dis-ease. New York: McGraw-Hill Book Com-pany, Inc., p. 667, 1966.
9. Rosenbloom, F. M., Henderson, J. F.,
Cald-well, I. C., Kelley, W. N., and Seegmiller,
J. E.: Biochemical bases of accelerated purine biosynthesis de novo in human fibroblasts
lacking hypoxanthineguanine
phosphoribo-syltransferase. J. Biol. Chem., 243:1166,
1968.
10. Tomkins, C. M., Garren, L. D., Howell, B. B.,
and Peterkofsky, B.: The regulation of en-zyme synthesis by steroid hormones: The role of translation. J. Cell. Comp. Physiol.
(Suppl. 1)66:137, 1965.
11. Loeb, J. N., Howell, R. B., and Tomkins, C. M.:
Turnover of ribosomal RNA in rat liver.
Science, 149:1093, 1965.
12. Jacob, F., and Monod, J.: On the regulation of gene activity. Sympos. Quant. Biol., 26:193,
1961.
13. Levinthal, C., Fan, P. D., Higa, A., and Zim-merman, B. A.: The decay and protection of
messenger RNA in bacteria. Sympos. Qaunt.
Biol., 28:1, 1963.
14. Revel, M., and Hiatt, H. H.: The stability of
liver messenger RNA. Proc. Nat. Acad. Sd.
U.S.A., 51:810, 1964.
15. Villa-Trevino, S., Farber, E., Staehlin, T., Wettstein, F. 0., and Noll, H.: Breakdown
and reassembly of rat liver ergosomes after
administration of ethionine of puromycin. J. Biol. Chem., 239:3826, 1964.
16. Carren, L. D., Howell, B. B., Tomkins, C. M.,
and Crocco, R. M.: A paradoxical effect of
actinomvcin D: The mechanism of regu-lation of enzyme synthesis by
hydrocorti-sone. Proc. Nat. Acad. Sci. U.S.A., 52:1121,
1964.
17. Thompson, E. B., Tomkins, C. M., and Curran,
J. F.: Induction of tyrosine-a-ketoglutarate
transaminase by steroid hormones in a newly established tissue culture cell line. Proc. Nat.
Acad. Sci. U.S.A., 56:296, 1966.
18. Schimke, B. T., Sweeney, E. W., and Berlin,
C. M.: Synthesis and degradation of rat liver
tryptophan pyrrolase. J. Biol. Chem., 240:
322, 1965.
19. Helinski, D. B., and Yanofsky, C.: Genetic
control of protein structure. In Neurath, H.,
ed.: The Proteins, Vol. 4. New York and
London: Academic Press, p. 1, 1966.
20. Siniscalco, M., Klinger, H. P., Eagle, H.,
Ko-prowski, H., Fujimoto, W. Y., and
Seegmil-ler, J. E.: Evidence for intergenic
comple-mentation in hybrid cells derived from two
human diploid strains each carrying an X-linked mutation. Proc. Nat. Acad. Sci. U.S.A., 62:793, 1969.
21. Yaffe, S. J., Levy, C., Matsuzawa, T., and
Ba-liah, T.: Enhancement of
glucuronide-conju-gating capacity in a hyperbilirubinemic
in-fant due to apparent enzyme induction by
phenobarbital. New Eng. J. Med., 275:1461, 1966.
22. Crigler, J. F., Jr., and Cold, N. I.: Effect of so-dium phenobarbital on bilirubin metabolism
in an infant with congenital, non-hemolytic,
