MORE ON ENZYMES

JULIUS P. MARIO, RMT, MS

 ENZYME REGULATION

 Feedback Control
 Proenzymes
 Allosterism
 Protein Modification
 Isoenzymes
 FEEDBACK CONTROL
 Formation of the product inhibits an earlier
reaction in a sequence

 In the reaction, E1 E2 E3

ABCD
 D may inhibit E1 activity by competitive or
noncompetitive inhibition
 When D is low, all three reactions proceed rapidly
 When D is high, E1 becomes inhibited
 PROENZYMES
 Inactive form of an enzyme
 Also known as zymogen
 Activated by cleavage of excess polypeptide
chain
 Cleavage of peptide promotes structural
changes, thus, functional
 Examples are
 Trypsinogen = cleavage of the 6 amino acids
from the N-terminal by enteropeptidase converts it
to active trypsin
 Angiotensinogen = cleavage of the last 2 amino
acids from the C-terminal by ACE converts it to a
vasoactive octapeptide, angiotensin
 PROENZYMES
 Chymotrypsinogen = cleavage of the 245
residues long polypeptide between R-15 and
I-16 from the N-terminal by trypsin converts it
to active п-chymotrypsin but the fully active
form is α-chymotrypsin
 PROENZYMES
 Some enzymes have proenzymes because
they are highly destructive when produced
directly in their active forms

 Should these leak or exist in excess,

inhibitory proteins bind them to render
them inactive
 ALLOSTERISM
 If a substance binds noncovalently and
reversibly to a site other than the active
site,
 It may inhibit the enzyme
(Negative modulation)
 It may stimulate enzyme action
(Positive modulation)
 ALLOSTERISM
 Allo means other; steric means shape
 Possible conformational changes affect the
behavior of proteins
 Due to multiple forms of the quaternary
structure of some allosteric enzymes
 A substance that modifies 4o structure and
thus, affect behavior is an allosteric effector
 Allosteric effectors are substrates,
activators, and inhibitors
 ALLOSTERIC PROTEINS
 Proteins in which subtle changes at one site
affects the structure and function of
another site

 Due to cooperative effects

 Depicted as sigmoidal curve on enzyme

kinetics

 Examples are Aspartate transcarbamoylase

(ATCase) & Hemoglobin
NONALLOSTERIC PROTEINS
 At first, dependent on the [S] with a
maximal rate not dependent on [S]
anymore

 Depicted as a hyperbolic curve on

Michaelis-Menten kinetics

 Examples are chymotrypsin & myoglobin

HOMOTROPIC vs. HETEROTROPIC
Effects
 Homotropic effects are allosteric
interactions that occur when several
identical molecules are bound to a
protein.

 Heterotropic effects are allosteric

interactions that occur when different
substances are bound to a protein.
 PROTEIN MODIFICATION
 Modification is usually a change in the
primary structure, typically by the addition
of a functional group covalently bound to
the apoenzyme

 Best-known example is the

activation/inhibition of phosphorylation
 Glycogen phosphorylase is active when it is
phosphorylated at its serine or tyrosine
residue
 Pyruvate kinase from the liver is inactive when
it is phosphorylated
 ISOENZYMES
 Enzymes that perform the same function
but have different combinations of
subunits, thus have different 4o structures

 Have different electrophoretic mobilities,

Km, and origins
 Act on the same substrate

 LDH has 4 subunits

 LD1(H4), LD2 (H3M), LD3 (H2M2), LD4
(HM3) & LD5 (M4)
 CARBOXYPEPTIDASES
 Cleave peptides and proteins at the carboxyl
terminal of a particular amino acid in the
chain

= at lysyl or arginyl
 Trypsin
 Chymotrypsin = at phenylalanyl,
tryptophanyl or tyrosyl as well
as L, H & Q
 Pepsin= same with chymotrypsin and others
 Thermolysin = at isoleucyl, leucyl or valyl
 PROTEASE FAMILY
 All members have similar chemical form

 Serine Proteases = cleaves at seryl residues

(chymotrypsin, trypsin and elastin)

 Cysteine protease = cleaves at cysteinyl residue

(papain, a meat tenderizer)

 Aspartyl protease = a pair of aspartate side

chains, sometimes on different subunits
participate in the reaction (pepsin and HIV
protease)
 ABZYMES
 Antibodies elicited by antigenic proteins
 Designer enzymes which can catalyze a
wide variety of reactions
 Usually are transition-state analogs which
when introduced into the body becomes
immunogenic
 Nα-(5’-phosphopyridoxyl)-L-lysine is
the transition-state analog for the reaction
between an amino acid and pyridoxal-5’-
phosphate
 CELLULAR LEVEL
 SER enzymes
 Detoxification by the cytochrome P-450
enzymes
 Lipid synthesis & degradation by
 Cytosolic side = cyt b5, cyt b5 reductase,
NADPH-ferrihemoprotein reductase, ATPase, 5’-
NT, glycolipid mannosyl transferases,
hydroxymethylglutaryl-CoA reductase, some 30
enzymes for steroid synthesis and 20 enzymes
of lipid synthesis
 Luminal side = cyt P-450, cyt P-450 reductase,
glucose-6-phosphatase and β-glucuronidase
 RER
 Transportenzymes for glycosylation of newly
formed proteins; flippases for phospholipid
synthesis

 GOLGI BODY
 Enzymes for posttranslational modifications of
proteins synthesized on membranes as well as
recycling of membrane material; 5’-NT,
NADH:cyt c oxidoreductase, NADPH:cyt c
reductase, UDP-galactose-N-
acetylglucosamine-β-D-
galactosyltransferase, and many
glycosylation enzymes
 LYSOSOMES
enzymes such as proteinases,
 Hydrolytic
glycosidases, lipases, phosphatases,
nucleases, and sulfatases

 MICROBODIES
 Mainly oxidative enzymes

 VACUOLES
 V-type H+-translocating ATPase
 BRUSH BORDER ENZYMES
 Disaccharidases found on the microvilli of
the small intestine

 Once lost, can be re-synthesized via

enzyme induction

 Substrates commonly acted upon are

maltose, sucrose, and lactose
 ENZYMES AS VIRULENCE
FACTORS
 Helicobacter pylori survives the harsh
gastric milieu by the urease on its cell
wall. The ammonia produced counters the
harmful effects of the acid.

 Some pathogenic cocci are capable of

producing hemolysins which interfere
with oxygen transport in an organism
 EFFECTS ON ENZYMES
 Bactericidal antibiotics for both Gram
positive and Gram negative organisms act
via inhibition of cell wall synthesizing
protein synthase (e.g. Carbapenems)

 Binds to the β subunit of RNA

polymerase to inhibit transcription of
mostly Gram+ bacteria & Mycobacteria
(e.g. Rifampin)
 An example of a medicinal enzyme inhibitor is
sildenafil (Viagra), a common treatment for male
erectile dysfunction.

 This compound is a potent inhibitor of cGMP

specific phosphodiesterase type 5, the enzyme
that degrades the signalling molecule cyclic
guanosine monophosphate.

 This signalling molecule triggers smooth muscle

relaxation and allows blood flow into the corpus
cavernosum, which causes an erection. Since
the drug decreases the activity of the enzyme
that halts the signal, it makes this signal last for
a longer period of time.
 Another example of the structural similarity of
some inhibitors to the substrates of the enzymes
they target is seen in the figure comparing the
drug methotrexate to folic acid. Folic acid is the
oxidised form of the substrate of dihydrofolate
reductase, an enzyme that is potently inhibited
by methotrexate. Methotrexate blocks the action
of dihydrofolate reductase and thereby halts
thymidine biosynthesis. This block of nucleotide
biosynthesis is selectively toxic to rapidly
growing cells, therefore methotrexate is often
used in cancer chemotherapy.
 Drugs also are used to inhibit enzymes
needed for the survival of pathogens. For
example, bacteria are surrounded by a
thick cell wall made of a net-like polymer
called peptidoglycan. Many antibiotics
such as penicillin and vancomycin inhibit
the enzymes (the transpeptidase from the
bacteria Streptomyces R61) that produce
and then cross-link the strands of this
polymer together. This causes the cell wall
to lose strength and the bacteria to burst.
 Drug design is facilitated when an enzyme
that is essential to the pathogen's survival
is absent or very different in humans.

 Humans do not make peptidoglycan,

therefore inhibitors of this process are
selectively toxic to bacteria.

 Selective toxicity is also produced in

antibiotics by exploiting differences in the
structure of the ribosomes in bacteria, or
how they make fatty acids
 AcCHE, an enzyme found in animals from insects to
humans. It is essential to nerve cell function through its
mechanism of breaking down the neurotransmitter
acetylcholine into its constituents, acetate and choline.

 This is somewhat unique among neurotransmitters as

most, including serotonin, dopamine, and
norepinephrine, are absorbed from the synaptic cleft
rather than cleaved.

 Reversible competitive inhibitors, such as

edrophonium, physostigmine, and neostigmine, are used
in the treatment of myasthenia gravis and in
anaesthesia. The carbamate pesticides are also
examples of reversible AChE inhibitors.
 The organophosphate insecticides such as malathion,
parathion, and chlorpyrifos irreversibly inhibit
acetylcholinesterase.
ENZYME DEFICIENCIES
 MUCOPOLYSACCHARIDOSIS
MPS Type Eponym Deficient Enzyme
IH Hurler a-iduronidase
I H/S Hurler-Scheie a-iduronidase
IS Scheie a-iduronidase
II Hunter Iduronidase sulfatase
III‡ Sanfilippo A Heparan sulfatase
Sanfilippo B N-acetylglucosaminidase
Sanfilippo C Acetyl CoA glucosamine
acetyltransferase
Sanfilippo D N-acetylglucosamine-6-sulfatase
IV Morquio A Galactosamine-6-sulfatase
Morquio B b-galactosidase
V Nonexistent
VI Maroteaux-Lamy N-acetylhexosamine-4-sulfatase
VII Sly b-glucuronidase
IX Hyaluronidase Hyaluronidase
Deficiency
GLYCOGEN STORAGE DISEASES
Type - Enzyme Deficient -Clinical Features
I (von Gierke) - Liver and kidney Glucose-6-phosphatase
Hepatomegaly, lactic acidosis, hyperlipidemia, severe fasting
hypoglycemia
II (Pompe)- All tissues’ alpha 1,4-glucosidase
Cardiomegaly, muscle weakness, death in infancy and adults
III (Cori-Forbes)- All tissues’ debrancher enzyme
Hepatomgaly, muscle weakness, fasting hypoglycermia
IV (Andersen)- All tissues’ brancher enzyme
Portal cirrhosis, death in infancy
V (McArdle)- Muscle phosphorylase
Pain and stiffness after exertion; myoglobinuria
VI (Hers) Liver phosphorylase Hepatomegaly,
mild fasting
hypoglycemia

VII (Tarui) Muscle and liver Pain and stiffness

phosphofructokinase on exertion
VIII Brain and liver Spasticity,
adenylate kinase decerebration,
high urinary
catecholamines,
death in infancy
IX Liver phosphorylase Hepatomegaly,
kinase occasional fasting
hypoglycemia
X Liver and muscle cAMP- Hepatomegaly
dependent kinase only
 LIPIDOSES
a group of inherited disorders
characterized by the
accumulation of lipids in
tissues especially the brain
due to
deficiency in a particular
sphingolipid catabolic enzyme
Niemann-Pick disease
 Deficiency in sphingomyelinase and
accumulation of sphingomyelin

Gaucher’s disease
 Deficiency in -D-glucosidase and
accumulation of glucocerebroside
Krabbe’s disease
 Deficiency in -D-galactosidase and
accumulation of galactocerebsides

Fabry’s disease
 Deficiency in -D-galactosidase and
accumulation of ceramide trihexoside
Tay-Sach’s disease
 Deficiency in -D-hexaminidase A
and accumulation of ganglioside GM2

Metachromatic Leukodystrophy
 Deficiency in sulfatide sulfatase and
accumulation of -
sulfogalactocerebroside