Clinical Science and Molecular Medicine (1977)53, 197-203.
EDITORIAL REVIEW
-Defectsof enzyme regulation in metabolic disease
?I,
I
D. J. GALTON, D. J. BETTERIDGE, K. G. TAYLOR,
G. HOLDSWORTH A N D J. STOCKS
Diabetes and Lipid Research Laboratory, St Bartholomew’s Hospital, London
Key words: enzymes, errors of metabolism,
metabolic disease, regulation.
Introduction
It is becoming clear that the Jacob-Monodmodel
for the regulation of enzyme activity in bacteria
(Fig. 1) is also the basis for enzyme regulation
in eukaryotic cells (Newsholme & Start, 1973),
although some components of the system, such
as repressors, have not yet been isolated from
mammalian cells. It is also probable that the
regulation of transcription of DNA is more
complex than that depicted in Fig. 1. However,
examples of regulation of enzyme activity by
induction or repression, allosteric modification
or covalent activation of enzymes have all been
identified in human cells (Galton, 1971). Three
types of enzyme regulation can therefore be
recognized from the Figure.
(a)Primary regulatory mechanisms. These are
responsible for the allosteric transition of enzymes between active and inactive states. They
are fast-acting mechanisms, occurring over
seconds, and exert a ‘fine’ control over metabolic pathways.
(b) Secondary regulatory mechanisms. Catalytic activity is governed by covalent modification of enzymes often involving phosphorylation or dephosphorylation reactions of seryl
residues which may alter the polymeric state of
the protein. Enzymes are activated or inhibited
at slower rates, over minutes, compared with
allostericmodulations. Covalent modification of
enzymes can alter the subunit structure of the
protein, and the change in the structure results
Correspondence: Dr D.J. Galton, Diabetes and Lipid
Research Laboratory, St Bartholomew’s Hospital,
London, E.C.1.
in an alteration of catalytic activity. Again this
mechanism is used for ‘fine’control of metabolic
processess and often involves the action of
hormones on metabolic pathways, e.g. the activation of lipolysis by catecholamines.
(c) Tertiary regulatory mechanisms. The activity of enzymes can be governed by alteration
in the rates of synthesis (or degradation) of
enzymic protein. This is a slow set of reactions
for human cells (hours to days) and is mediated
by the transcription of DNA and synthesis
de nouo directed by mRNA. Enzyme synthesis in mammalian cells can be induced or
repressed by metabolites (or hormones) as in
bacterial cells. Examples are the induction of
tyrosine aminotransferaseby corticosteroidhormones in rat hepatocytes (Tomkins, 1967) and
the repression of hydroxymethylglutaryl-CoA
reductase by low-density lipoprotein in human
fibroblasts (Brown & Goldstein, 1976). Such
mechanisms provide a long-term or ‘coarse’
control of metabolic pathways on which the
other two mechanisms are superimposed. Thus
for any one metabolic pathway it is likely that
all three mechanisms will be involved in its
regulation (Scrutton & Schramm, 1977).
It is now established that defects can occur in
the regulation of enzymic activity and give rise
to a different type of pathology than the classical
inborn errors of metabolism (Galton, Higgins &
Reckless, 1975). The classical inborn errors are
individually rare disorders in which the catalytic activity of enzymes or the transport function of proteins is impaired. The defect is
usually a mutation in a structural gene and an
amino acid substitution affecting the active site
of the corresponding protein. They are rare
disorders as evolutionary selection is usually
against their appearance in the population and
197
D. J. Galton et al.
198
+Regulator
gene+
loperator g e n e t s t r u c t u r a l gene+
4
DNA
1
Inducer4
I
Activatic
CYJk
6y < Repressor
Active enzyme
F
Substrate + Cofactor
FEEDBACK INH18ITION
Fro. 1. Scheme for the regulation of enzyme activity.
affectedindividuals often fail to reach the reproductive period. One of the commonest inborn
errors, phenylketonuria, occurs in the population
at a frequency of between 1 :3000 and 1:lo7
(depending on locality). In general their effects
on metabolic pathways lead to an abnormal accumulation of intermediates, e.g. phenylpyruvic
acid, in body fluids and to a reduction in the
supply of the terminal product of the pathway.
Frequently the intermediary metabolites that
accumulate can damage tissues and lead to the
pathological features of the disease.
In contrast, defects in enzyme regulation can
lead to a loss of modulation of metabolic pathways with a consequent overproduction of the
terminal product without gross accumulation of
intermediary metabolites. A mutation affecting
an allosteric site of an enzyme without affecting
its catalytic site might allow the enzyme to
function normally as a catalyst but still impair
the response of the enzyme to allosteric modifiers. If such an abnormal enzyme catalysed a
rate-determining step in a biosynthetic pathway,
unregulated synthesis of the end product might
occur. Since the terminal products of pathways
(e.g. glucose, triglyceride, uric acid) are often
less harmful than metabolic intermediates there
may be less evolutionary selection against
their appearance, and the frequency of such
disorders could be several orders of magnitude
greater than the classical inborn errors. Also
the therapeutic implications of the two groups
of metabolic defects are quite different (Galton
& Betteridge, 1977). The definitive treatment
of the inborn errors is by enzyme replacement,
which is theoretically possible but still a long
way from practical use. On the other hand,
regulatory defects of enzymes might be treated
more easily because affected tissues already
contain a catalytically active protein. The
simplest case would be loss of allosteric inhibitory sites on an enzyme or a defective repressor,
both of which would lead to enhanced catalytic activity of the enzyme. A specific enzyme
inhibitor would then be appropriate therapy
and such inhibitors are already in use in other
branches of clinical medicine (e.g. allopurinol).
Conversely a defect of allosteric stimulatory
sites on an enzyme would produce a clinical
;picture resembling an inborn error except
for the presence in tissues of a catalytically
active protein but maintained in an inhibited
state by the 'function of unopposed inhibitory
sites on the enzyme. Appropriate therapy would
be analogues of the enzyme stimulator that can
combine with the deformed allosteric site and be
used pharmaceutically to restore activity of the
enzyme. In this context it may be important
Enzyme regulation defects
to study all the described inborn errors of
metabolism to see if they could be due to
defects of allosteric stimulatory sites rather
than defects of catalytic sites. Therapy would
be much simpler if the former were true.
Finally, knowledge of physiological agents
(hormones or metabolites) which induce
or repress synthesis of proteins may allow
therapy of unregulated enzyme activity by
modifying synthesis at the level of the genome.
A preliminary stage in this direction is the use
of phenobarbitone to induce hepatic uridine
diphosphate glucuronyl transferase so as
to enhance the metabolism of bilirubin in
cases of neonatal hyperbilirubinaemia. A
summary of the differences between regulatory
and catalytic enzymic defects is presented in
Table 1.
In clinical medicine defects probably occur
in the regulatory as well as in catalytic activity
of enzymes. They can be conveniently classified
by the level at which the regulatory defect
occurs. Thus one can recognize primary,
secondary and tertiary disorders of enzyme
regulation giving rise to a variety of pathological
conditions. However, their identification is
a fairly new development and the concept
that they contribute to disease processes
may in some casesbecontroversial.Theevidence
for the occurrence of each type will now be
reviewed briefly.
P r i i regulatory defects
Lipomtosis with defective regulation of phosphofructokinase
6-Phosphofructokinase(EC.2.7.1.1 1)is a r a t e
determining enzyme catalysing the phosphorylation of fructose &phosphate to fructose
1,6-diphosphate and the entry of glycosyl
units into glycolysis. In the human adipocyte
the major fate of glycosyl units is their conversion
into glyceride glycerol for storageas triglyceride,
very little being converted into long-chain
fatty acids. The source of fatty acyl groups
for storage in adipose tissue is mainly the
liver, and they are transported on very-lowdensity lipoproteins to adipose tissue for
esterification with glycerol phosphate.
Many modifiers are known to affect the
activity of phosphofructokinase in adipose
tissue; the enzyme is stimulated by fructose
199
‘
6-phosphate, AMP and ammonium ions.
It is inhibited by high concentrations of ATP
(>2 mmolll) and also by citrate. The physiological role of all these modifiers is not clearly
understood and some may be artifacts of
an assay system in vitro. The rates of glycolysis
in perfused rat heart are inversely related
to the tissue amounts of citrate (Randle, 1966)
but there is still no direct evidence that citrate
is an important regulatory factor in the intact
animal. Citrate is known to affect the activity
of phosphofructokinase in adipose tissue in
much the same way as in skeletal or cardiac
muscle (Denton & Randle, 1966), but a role
for the regulation of glycolysis by citrate
is more controversial in adipose tissue, since
amounts of citrate do not appear to relate to
changes in gIycoIytic flux.
Lipomatosis is a condition characterized
by excessive synthesis and accumulation of
triglyceride by a clone of adipocytes, which
results in ‘fatty’ tumours anywhere in the body.
It is possible that the metabolic control
relating to triglyceride metabolism in these
tumours is defective. The activity and hormonal
control of lipolysis and lipogenesis in lipomata
does not differ from metabolic pathways
in adjacent adipose tissue. The only metabolic
lesion that has been demonstrated in such
lipomatais a defect in theregulationof glycolysis.
In extracts of lipomata the pathway appears
to be insensitive to inhibition by citrate,
suggesting defective allosteric modification
of the enzyme. When the enzyme is extracted
from normal adipose tissue and lipomata
respectively and a kinetic analysis performed,
enzymes from both sources are found to be
sensitive to inhibition by high concentrations
of ATP, but the tumour enzyme appears
to have lost sensitivity to inhibition by citrate
(Atkinson, Galton & Gilbert, 1974). Thus
citrate (5 mmol/l) inhibits the normal enzyme
by approximately 60%, whereas the tumour
enzyme is inhibited by approximately only
10%. The total extractable activity of phosphofructokinase in both tissues is similar
at 0.03 unitlmg of protein. The consequences
of such a lesion in vivo might result in loss
of regulation of glycolysis with regard to
tissue amounts of citrate, so that the pathway
might be fully active under all nutritional
conditions to favour the conversion of glycosyl
residues into glyceride glycerol for storage
D. J. Galton et al.
200
TABLE
1. Comparison of the classical inborn errors ef metabolism with defects in enzyme
regulation
Defect
Pathogenesis
Frequency
Effect on metabolic
product
Effect on metabolic
intermediates
Examples
Classical inborn errors of
metabolism
Defects in enzyme
regulation
At active site of enzyme or
carrier protein or absence
of enzyme
Inherited
1 :I0000for
phenylketonuria
Depletion
At regulatory site(s) of enzyme
or carrier protein
Accumulation
No gross changes
Phenylketonuria,
homocystinuria
Lipomatosis.
familial hypercholesterolaemia
as triglyceride. This may partly account for
the excessive storage of triglyceride in these
tumours, although it is unlikely to account
for the initial development of such lesions.
From animal studies defective regulation
of phosphofructokinase has been shown to
arise on a genetic basis. A mutant strain of
mouse (Bar Harbor, strain C57BL/6J, ob/ob)
has been described that develops obesity,
hyperinsulinaemia and hyperlipaemia inherited
as a simple Mendelian recessive. The homozygote is severely affected whereas heterozygous littermates are difficult to distinguish
from control animals.
The metabolic lesion underlying this condition
has not been elucidated, but it presumably
must be expressed as a mutant protein. The
metabolic activity of the adipose organ of
affected animals appears to be normal. However,
an abnormality in the regulation of phosphofructokinase isolated from the liver of affected
animals has been reported by Katyare &
Howland (1974). These kinetic studies of the
partially purified hepatic enzyme from affected
animals have shown defective inhibition by
citrate which resembles the observed defect
in lipomatosis. In addition the enzyme from
the oblob mouse has decreased sensitivity to
inhibition by high concentrations of ATP,
suggesting that two allosteric sites may be
defective in this enzyme.
The mutation is associated with both excessive
production of triglyceride in the liver and
excessive storage in the adipose organ. Again
it is possible that the lesion described by
Inherited or acquired
1 :lo0 for lipomatosis
Accumulation
Katyare & Howland (1 974) could contribute
to excess production of glyceride glycerol
by failing to modulate the entry of glycosyl
units into the biosynthetic pathways under
conditions of fasting and feeding. Thus the
pathway would be fully active under conditions such as fasting, which would normally
be expected to inhibit glyceride glycerol
synthesis; and this may partly contribute to
the excess deposition of triglyceride found in
this condition.
The defective regulation of phosphofructokinase in lipomatosis is associated with a change
in pool size of the end product in vivo (tissue
triglycerides) in the expected direction of the
unregulated pathway. This provides indirect
evidence that the allosteric defect could have
physiological consequences in vivo. It is difficult
to gain more direct evidence for a role of
an allosteric defect in vivo other than parallel
changes between the accumulation of metabolic
products in the intact animal and an unregulated
pathway in vitro which would be expected to
lead to this. The final test must come from the
use of speci6c enzyme inhibitors in vivo and
if these produce a corresponding decline in
pool size of the terminal product, then this is
additional evidence that the enzymic lesion
does in fact have pathophysiological consequences.
Gout with excessive purine production
Phosphoribosylpyrophosphate (PRPP) synthetase (EC 2.7.6.1) catalyses the formation of
Enzyme regulation defects
PRPP from ribose 5-phosphate and ATP.
The enzyme is activated by inorganic phosphate
and inhibited by compounds such as ADP and
GDP. PRPP is a substrate for the first uniquely
committed enzyme in the synthesis of purine
nucleotides de novo and is an important regulator of the pathway. It has been proposed that
increased PRPP availability could account
for accelerated purine synthesis de novo in
some types of gout; a new variant of gout
associated with excessive purine production
and uric acid lithiasis has been reported
(Zoref, de Vries & Sperling, 1975). PRPP
synthetase in erythrocytes of two affected
siblings exhibited a defect in the feedback
inhibition of the enzyme to GDP and ADP,
resulting in an increased PRPP content of
cells and availability for nucleotide synthesis.
Cultured human fibroblasts, unlike erythrocytes,
possess the complete pathways for purine
metabolism except for xanthine oxidase (Kelley
& Wyngaarden, 1970). When skin fibroblasts
from affected members of the family were
cultured, PRPP synthetase in dialysed lysates
of fibroblasts showed a decreased sensitivity
to inhibition by ADP and GDP. The PRPP
content and rate of purine synthesis de novo
were markedly increased in fibroblasts of
affected members, but were normal in other
family members.
This lesion differs from the increased availability of PRPP in cells of patients with the
Lesch-Nyhan syndrome with decreased utilization of PRPP due to a phosphoribosyltransferase defect. Two additional families
with gout associated with increased PRPP
synthetase activity have subsequently been
described (Becker, Meyer, Wood & Seegmiller,
1973).
Secondary regulatory defects
Triglyceride storage disease with defective
activation of triglyceride lipase
Triglyceride lipase (EC 3.1.1.3) is the ratedetermining enzyme for the breakdown of
triglyceride in the human adipocyte. It is of
especial interest because it is under hormonal
control mediated by catecholamines, cyclic
AMP and protein kinase. Conversion of the
201
inactive into the active form of triglyceride
lipase involves a phosphorylation of seryl
residues on the enzyme (Huttenen & Steinberg,
1971), and a defect in the activation of triglyceride lipase would be expected to lead
to a decrease in triglyceride catabolism in
adipocytes.
Two children have been recently reported
with a defect in the activation of lipolysis by
catecholamines in peripheral adipocytes (Galton, Reckless & Taitz, 1976). This was associated with abnormal storage of triglyceride
in peripheral adipose tissue becoming evident
when the children failed to thrive. This caused
depletion of truncal deposits of adipose
tissue but did not mobilize triglyceride from
peripheral deposits. Metabolic studies of
peripheral adipose tissue from one of the children
revealed normal rates of triglyceride synthesis
from exogenous glucose and palmitate, compared with truncal tissue, but after incubation
of the tissue with isoprenaline
mmol/l)
there was failure of activation of lipolysis,
although basal activities of the enzyme were
normal (Galton, Gilbert, Lucey & Walker-Smith,
1977). It is possible that the failure of activation
of triglyceride lipase is related to the abnormal
storage of triglyceride in peripheral adipocytes.
Glycogenstorage disease with defective activation
of phosphorylase b
Variants of glycogen storage disease (type
VIII) have been described where there is
a defect in the protein kinase activation of
phosphorylase in the liver. Hug, Schubert
& Chuck (1970) reported on patients with a
defect of protein kinase involved in the activation of phosphorylase b and an accumulation
of glycogen in liver and muscle. Homogenates
of patients' muscle lacked a cyclic-AMPdependent protein kinase and were unable
to activate phosphorylase b of rabbit muscle
except at a tenth of the normal rate. Activation
of phosphorylaseb could be restored by addition
of mouse muscle homogenates containing
protein kinase but deficient in phosphorylase
kinase. This therefore indicates a reduction
in protein kinase activity in the patients'
muscles and a failure of activation of phosphorylase, which may account for the excess
deposition of glycogen in their tissues.
202
D. J. Galton ef a[.
Tertiary regulatory defects
Familial hypercholesterolaemia and defective
regulation of hydroxymethylglutaryl-CoA reductase
Hydroxymethylglutaryl (HMG)-CoA reductase (EC 1.1.1.34) is an important rate-determining enzyme on the pathway for cholesterol
biosynthesis. It catalysestheconversion ofHMGCoA into mevalonate, which is the first committed step for sterol synthesis in many cells.
Themechanismsgoverningthe activity of HMGCoA reductase are complex and in many
cell types (fibroblasts, leucocytes, lymphocytes) involve a specific high-affinity cellsurface receptor for combination with lowdensity lipoprotein (LDL) (Brown & Goldstein,
1976). After combination with cell receptor
there is repression of the enzyme HMG-CoA
reductase, although the mechanism for this
is not fully understood.
Familial hypercholesterolaemia is an inherited disorder of lipoprotein metabolism due
to anautosomal dominant mutation. It is characterized by accumulation of LDL in plasma,
xanthomatous lipid deposits in skin and
tendons and the development of premature
arterial disease. Recent studies with cultured
fibroblasts from subjects with the disease
have shown a defect in the repression of
HMG-CoA reductase by LDL. Brown &
Goldstein (1976) propose that the primary
metabolic abnormality is an absence or impairment of high-affinity cell receptors for combination with LDL-apoprotein, which results
in defective LDL catabolism as well as loss
of regulation of cholesterol biosynthesis.
It is likely that the former accounts for the
accumulation of LDL in plasma and its
reduced fractional catabolic rate, whereas
the loss of regulation of HMG-CoA reductase
is phenotypically linked to the cell receptor
defect.
Studies of patients with familial hypercholesterolaemia suggest that it may be a
genetically heterogeneous group of disorders.
Another possible mechanism for defective
regulation of HMG-CoA reductase and LDL
catabolism is on the LDL at sites for combination
with cell receptor. A kindred has been observed
(Higgins, Lecamwasam & Galton, 1975) where
the proband, a sibling and daughter pos-
sessed a LDL that failed to suppress the
activity of HMG-CoA reductase in normal
leucocytes, whereas the enzyme in the proband's leucocytes was suppressed by LDL
obtained from control subjects, indicating
that there was no cellular defect in the regulation of HMG-CoA reductase
The abnormality of the LDL did not alter
its fractional catabolic rate when injected
into a normal subject, nor did it alter its
binding properties to normal lymphocytes
(Myant, Reichl, Thompson, Higgins & Galton,
1976).
It is possible that in this variety of familial
hypercholesterolaemia the defect in regulation
of HMG-CoA reductase is of greater aetiological significance than the reduced fractional
catabolic rate of the lipoprotein and may be
responsible for cellular accumulation of cholesterol without a marked increase in plasma
amounts of LDL. Such cellular accumulation of
sterols might initiate the fatty streak of the
intimal lining and predispose to development
of atheroma. The proband died from a myocardial infarction at 36 years despite normal
plasma concentrations of cholesterol (4-3
mmol /l) for the previous 10 years.
Conclusion
The examples given above demonstrate the
occurrence of defects of enzyme regulation in
vitro, although their role in the development
of a disease state may be still controversial.
If they are of pathological significance in
vivo they would be expected to lead to a
different type of disorder than the classical
inborn errors of metabolism.
Common metabolic diseases such as adult
diabetes, the hyperlipidaemias and some types
of obesity are all associated with accumulation
of normal metabolic end products (glucose,
triglyceride, cholesterol) and they may be due
to disorders of enzyme regulation.
It should be emphasized that the examples
of defective enzyme regulation that have been
discussed represent only the first steps in
the application of principles of molecular
biology to our understanding of metabolic
disease. If other examples of defects in enzyme
regulation are observed as aetiological factors in
metabolic disease then they offer the exciting
therapeutic possibilities of developing specific
Enzyme regulation defects
enzyme inhibitors (or activators) to modulate
the unregulated metabolic pathways and
possibly improve the pathologicalconsequences.
Acknowledgments
Support for the work described in this paper
came from the Medical Research Council,
British Diabetic Association and the Joint
Research Board of St Bartholomew’s Hospital.
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