UNIVERSITÄTSMEDIZIN

NEUMARKT A. M. https://edu.umch.de
www.umfst.ro

CAMPUS HAMBURG PRESENTER:

Prof Amelia TERO-VESCAN
Lecture 5. Enzymes
Lecture 5. Enzymes. 11. Regulation of enzyme activity
11.1. Allosteric enzymes
1. General considerations. 11.2. Covalent Modification
2. Enzyme proprieties 12. Isozymes (Isoenzymes)
13. Localization of enzymes and their origin in plasma
3. Enzyme nomenclature and classification
4. Enzyme specificity.
5. Enzyme Structure.
5.1. Inorganic ions as enzyme cofactor
6. Active site of enzyme
7. Catalytic mechanism of enzymes
8. Models of formation of the ES complex
9. Kinetics of enzymatic reactions
9.1. Factors that influence the rate of enzymatic reactions
10. Enzymatic effectors
10.1. Enzyme activators
10.2. Enzyme inhibitors

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11. Regulation of enzyme activity

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 The regulation of the enzymatic activity is achieved through 2 general mechanisms:

Changes in the catalytic efficiency

Quantitative modifications
• are achieved through the regulation enzymes which are
• are performed by the general mechanism of regulation of
of two types:
protein biosynthesis.
11.1. Allosteric enzymes, whose catalytic activity is
• Enzymes present the phenomenon of adaptation
modulated by non-covalent binding of a specific
• the intracellular concentration of a metabolite may increase
metabolite to a center on the surface of the enzyme,
or decrease by induction or repression the enzyme
different from the catalytic one;
biosynthesis.
11.2. Covalently modulated enzymes, which suffer in
• Enzyme induction and repression are controlled and
the presence of other enzymes, an interconversion
regulated by the nervous system and the hormonal system.
into an active or inactive form.

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11.1. Allosteric enzymes

Allosteric enzymes function through reversible, noncovalent binding of regulatory compounds called allosteric modulators
or allosteric effectors, which are generally small metabolites or cofactors

• Allosteric enzymes change conformation by the binding modulators, becoming more/less reactive
• Often the modulator is the substrate itself; regulatory enzymes for which substrate and modulator are identical are called
homotropic.
• The effect is similar to that of O2 binding to hemoglobin: binding of the ligand—or substrate, in the case of enzymes—
causes conformational changes that affect the subsequent activity of other sites on the protein.
• When the modulator is a molecule other than the substrate, the enzyme is said to be heterotropic
• !!! Alosteric modulators should not be confused with non-competitive inhibitors (although NCI bind at a second site on the
enzyme, they do not necessarily mediate conformational changes between active and inactive forms, and the kinetic
effects are distinct)

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Allosteric enzymes - Properties

• are significantly different from simple nonregulatory enzymes.

• in addition to active sites, allosteric enzymes generally have one or more regulatory, or allosteric, sites for binding the
modulator
• Just as an enzyme’s active site is specific for its substrate, each regulatory site is specific for its modulator.
• Enzymes with several modulators generally have different specific binding sites for each.
• In homotropic enzymes, the active site and regulatory site are the same.
• Allosteric enzymes are generally larger and more complex than nonallosteric enzymes.
• Most have two or more subunits.
• E.g. Aspartate transcarbamoylase, catalyzes one reaction in the biosynthesis of pyrimidine nucleotides has 12 polypeptide
chains

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Allosteric enzymes – Feed-back

• Some allosteric enzymes are specifically inhibited by the end product of the pathway
when the concentration of the end product exceeds = feedback inhibition.
• E.g. the bacterial enzyme system that catalyzes the conversion of L-threonine to L-
isoleucine in five steps
• the first enzyme, threonine dehydratase, is inhibited by isoleucine, the product of the last
reaction of the series.
• This is an example of heterotropic allosteric inhibition.
• Isoleucine is quite specific as an inhibitor as no other intermediate in this sequence
inhibits threonine dehydratase, nor is any other enzyme in the sequence inhibited by
isoleucine.
• Isoleucine binds not to the active site but to the regulatory site.
• This binding is noncovalent and readily reversible; if the isoleucine concentration
decreases, the rate of threonine dehydration increases

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Kinetic Properties of Allosteric Enzymes

• Allosteric enzymes kinetics is different from Michaelis-Menten kinetics.

• They do exhibit saturation with the substrate when [S] is sufficiently high, but for some allosteric enzymes, plots of V0
(reaction rate) versus [S] produce a sigmoid curve, rather than the hyperbolic curve.

• Sigmoid kinetic reflects cooperative interactions between

protein subunits, changes in the structure of one subunit are
translated into structural changes in adjacent subunits

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 E.g. Acetyl-CoA
Glucose

Gluconeogenesis
• in the mitochondria, pyruvate can be converted to acetyl-CoA (by the
pyruvate dehydrogenase) to start the Krebs cycle or oxaloacetate (by
Oxaloacetate
pyruvate carboxylase) to start gluconeogenesis
Pyruvate carboxylase
• When fatty acids are used as energy supply, there is an excess of acetyl-CoA
Pyruvate +
(from beta-oxidation of fatty acids)
Pyruvate dehydrogenase
• Acetyl-CoA is a positive allosteric modulator of pyruvate carboxylase and a - Acetyl-CoA
negative modulator of pyruvate dehydrogenase, so it stops glycolysis and
starts gluconeogenesis
Krebs cycle

Energy

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 11.2. Covalent Modification

• The covalent attachment of a molecule to an enzyme or protein can modify its activity.
• Most modifications are reversible.
• Phosphorylation and dephosphorylation are common means of covalent modification.
• The attachment of acetyl groups to lysine residues by acetyltransferases and their removal by deacetylases
• More than 2000 different proteins in mammalian cells regulated by acetylation
• Histones—proteins that are packaged with DNA into chromosomes—are extensively acetylated and deacetylated in vivo on
lysine residues

Modification Donor molecule Example of modified Protein function

protein
Phosphorylation ATP Glycogen phosphorylase Glucose homeostasis;
energy transduction
Acetylation Acetyl CoA Histones DNA packing;
transcription
gamma-Carboxylation HCO3- Thrombin Blood clotting 10
Enzyme phosphorylation
• 30% of eukaryotic proteins are phosphorylated.
• The enzymes catalyzing phosphorylation reactions are called protein kinases
• ATP is the most common donor of phosphoryl groups, and the terminal phosphoryl group of ATP is transferred to
a specific amino acid of the protein or enzyme.
• Transfers to Ser and Thr residues are handled by one class of protein kinases and to Tyr residues by another.

• The conversion of the non-charged polar OH

group to the –OPO32- polar form represents
a difference in structural information.
Active Protein kinase Inactive
• interactions with other vicinal lateral
residues are possible, which can cause
reorientations of remote groups with
conformational changes, reflected in the
activity modification: some enzymes are
Inactive
Active activated by phosphorylation, others are
Protein phosphatase 11
inactivated.
E.g. of enzymes activated or inhibited by phosphorylation

Enzyme phosphorilated active
Glycogen phosphorilase
Tissue lipase
Citrate lyase
Fructose-2,6-diphosphatase
Enzyme non-phosphorilated
active
Glycogen synthase
Pyruvate-dehydrogenase
Pyruvate kinase
Acetyl-CoA carboxylase

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 12. Isozymes (Isoenzymes)

Isozymes, or isoenzymes = enzymes that differ in amino acid sequence yet catalyze the same reaction.
• Typically, these enzymes display different kinetic parameters, such as km, or respond to different regulatory molecules
• They are encoded by different genes, which usually arise through gene duplication and divergence.
• Isozymes can often be distinguished from one another by physical properties such as electrophoretic mobility.

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E.g. lactate dehydrogenase (LDH), catalyzes a step in anaerobic glucose metabolism and glucose synthesis.
• 2 polypeptide chains for this enzyme:
• the H isozyme is highly expressed in heart muscle
• the M isozyme is expressed in skeletal muscle.
• The amino acid sequences are 75% identical and it is a tetrameric enzyme
• There are 2 types of peptide chains – M and H
• The H 4 isozyme, found in the heart, has a higher affinity for substrates than does the M 4 isozyme.
• The M 4 isozyme functions optimally in the anaerobic environment of hard-working skeletal muscle, whereas the H 4
isozyme does so in the aerobic environment of heart muscle.

The presence of some isozymes in the

blood is a sign of tissue damage
E.g. an increase in serum levels of H 4 is an
indication of heart attack

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E.g. Hexokinase,
• catalyzes the entry of free glucose into glycolysis
• 4 isozymes (designated I to IV)
• The predominant hexokinase isozyme of myocytes (hexokinase II) has a high affinity for glucose
• Muscle hexokinases I and II are allosterically inhibited by their product, glucose 6-phosphate, so when the cellular
concentration of glucose 6-phosphate increases, these isozymes are temporarily and reversibly inhibited
• predominant hexokinase isozyme of liver is hexokinase IV (glucokinase), with a high Km allows it to directly regulate the
blood glucose

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 E.g. Creatine kinase
Creatine kinase

ATP ADP

• CK catalyses a reversible reaction between creatine and ATP to create phosphocreatine (PCr) and ADP
• In tissues and cells that consume ATP rapidly, especially skeletal muscle, but also brain PCr serves as an energy reservoir

• CK enzymes consist of two subunits, which can be either B (brain type) or M (muscle type)
• There are, therefore, three different isoenzymes:
• CK-MM (muscle) muscular dystrophy
• CK-BB (brain)
• CK-MB (heart) – increases in myocardial infarction

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13. Localization of enzymes and their origin
in plasma

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1 the distribution of enzymes in different cells 2 Intracellular localization of different enzymes

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1 the distribution of enzymes in different cells
A

metabolic pathways that occur in all cells

Localization of the process it catalyzes Glycolysis Krebs cycle Protein nucleic acid
biosynthesis biosynthesis
The cells contain only those enzymes that
effectively intervene in the catalysis

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1 the distribution of enzymes in different cells
A

metabolic pathways that occur in all cells

Localization of the process it catalyzes B

metabolic pathways that take place in
specialized cells

Thyroid hormone
synthesis → Thyroid
gland

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 2 Intracellular localization of different enzymes

coincides with the localization of

the pathway or metabolic reaction
catalyzed by the respective enzyme
"de novo"
biosynthesis of
fatty acids
glycolysis
cytoplasm

cholesterol
biosynthesis Respiratory chain

mitochondria
-oxidation of fatty acids
Krebs cycle

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 2 Intracellular localization of different enzymes

enzymes with both cytoplasmic and

mitochondrial localization – AST,
malate-dehydrogenase

AST AST

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 2 Intracellular localization of different enzymes

respiratory chain
Lanţul respirator

ATP
ATP - synthase
sintetaza

respiratory chain enzymes

attached to the
mitochondrial membrane

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 Adrenaline, 2 Intracellular localization of different enzymes
INSULIN
Glucagon

ATP AMP
adenylate
cyclase Phosphodiesterase

cAMP protein kinase A

active
protein kinase A
enzymes included in the
inactive cytoplasmic membrane
E.g. adenylate cyclase

Regulatory Catalytic Regulatory

subunits subunits subunits Catalytic
subunits

Tissue lipase Tissue lipase

(-P) (+P)
inactive active

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 3 enzymes synthesized by specialized cells from different
organs, but are active outside the cells

enzymes involved in
food digestion

Pancreatic juice
enzymes
The exocrine
Pancreatic lipase pancreas

2-monoglycerids Free fatty acids

Amilase
pancreatică
Trypsin
Pancreatic lipase

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 Importance in diagnosis
• increased activity of mitochondrial localization enzymes suggests a more severe process, leading to mitochondrial
membrane destruction, whereas increased activity of cytoplasmic localization enzymes suggests less severe
injury, with changes in cell membrane permeability.
• it is used to determine the activity of plasma enzymes (for a correct interpretation it is necessary to know: the
place of production of the different enzymes, the mechanism by which they can reach the blood, their fate after
they are passed through the cells in the blood)

Functional plasma enzymes 2 Enzymes of exocrine

1 3
= enzymes secreted actively in
plasma (by liver) with
physiological role in plasma
E.g. coagulation enzymes
E.g. LCAT (lecithin-cholesterol acyl
transferase)
Liver disorders
Non-Functional Plasma Enzymes
secretions
= enzymes that diffuse passive into
= intracellular enzymes,
blood, with no role at this level
without a role in plasma
E.g. lipase, amylase, trypsin, etc
E.g. transaminases, creatin
kinase, lactate dehydrogenase.
etc.
Atrophy of the producing Increased permeability /
organs cell membrane damage
Increased permeability of
the producer cells
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