Enzymes are proteins that act on substrates, catalyzing chemical reactions within the cell.

Enzymes are specific in the sense that each enzyme only reacts with a few closely related
substrates. Some enzymes require cofactors (biotin, lipoamide, cobalamin) to function properly.
Enzymes can become denatured by changes in temperature or pH. Enzymes are classified as
oxidoreductases, transferases, hydrolases, lyases, ligases, and isomerases, based on the type of
reaction they catalyze. Enzyme kinetics is the study of enzyme reaction rates, which are
determined using the Michaelis-Menten and Lineweaver-Burk equations. These equations can be
also used to evaluate how different types of enzyme inhibitors affect the reaction rate. Enzyme
deficiencies can result in severe diseases such as Lesch-Nyhan syndrome, Gaucher disease, and
phenylketonuria.

Overview
Enzymes are complex proteins that catalyze chemical reactions. Enzymes act on substrates that
can either be cleaved or joined to form a new product (e.g., carbonic anhydrase enzyme → CO2
+ H20 ⇄ H2CO3). They are essential for life, if enzymes did not exist, cellular reactions would not
occur fast enough to sustain life. Thus, enzyme deficiencies can result in severe diseases (e.g.,
Lesch-Nyhan syndrome).
The name of enzymes is usually based on the reaction catalyzed plus the suffix -ase. For
example, the name of the enzyme that adds hydroxyl groups (OH-) formed as follows: hydroxyl
+ -ase → hydroxylase.

General characteristics of enzymes

 Active site: binding site for a specific substrate on a specific enzyme
 Specificity: Enzymes are highly specific for their substrate and product.
o Some exceptions include proteases that break down proteins to peptides in the
digestive system.

 Rate: enzymes catalyze reactions by a factor of 106–1011

 Coenzymes: many enzymes require coenzymes (e.g., biotin) that allow them to perform their
action on a substrate.

 Usually small organic molecules derived from metal ions or vitamins

 Thermodynamics
o Enzymes do not affect the energy level of substrates or products (free energy
released remains the same).
o Enzymes are able to decrease the energy of activation required to start a reaction.
o The velocity of enzymatic reactions increases with temperature (up to 37o C in
humans).
 Temperatures > 37o C slow down enzymatic reactions and can result in
denaturation of enzymes.
 pH: Each enzyme has a specific pH at which it can achieve maximum velocity (Vmax).
o Alterations in pH can cause denaturation of enzymes (specific to each enzyme).
  Example: Pepsin works best in acidic environment like the stomach (pH
∼1.5-2) and it is inactivated in the duodenum when bicarbonate is release
from the pancreas, increasing the pH to > 7.

Gibbs energy

Energy (∆G) for enzymatic reactions usually comes from the break down of ATP or GTP bonds
(hydrolysis). Enzymatic reactions can occur spontaneously or nonspontaneously. The following
are relationships between energy and enzymatic activity.

 Exergonic: Energy (∆G) < 0 → reactions can occur spontaneously (often irreversible)
 Endergonic: Energy (∆G) > 0 → reactions require energy to occur (from ATP or GDP)
 Balanced reaction: Energy (∆G) = 0 → the reaction is at equilibrium (reversible)

Classes of enzymes
Enzyme class Function Subclass Examples
 Dehydrogenases
 Examples: pyruvate
 Oxidases
dehydrogenase,
 Catalyze  Oxygenases
isocitrate
Oxidoreductases
redox  Hydroxylases
dehydrogenase,
reactions o Transfers
glucose-6-phosphate
hydroxyl dehydrogenase
groups (OH)

Transferases  Transfer  Kinases  Glucokinase,

functional o Transfer hexokinase,
groups phosphate phosphofructokinase,
groups from a pyruvate kinase
high energy  Glycogen
molecule phosphorylase
(e.g., ATP,
ADP) onto a
substrate
 Phosphorylases
o Add inorganic
phosphate
onto
substrates
o Do not
require any
energy source
 Aminotransferases
 Enzyme class Function Subclass Examples
 Glycosyltransferases

 Tyrosine
hydroxylase,
phenylalanine
hydroxylase,
tyrosine
hydroxylase

 Phosphatases
o Remove
 Cleave phosphate  Fructose-1,6-
covalent groups biphophatase,
bonds by
Hydrolases  Lipases glucose-6-
adding
 Peptidases phosphatase
water
 Nucleosidases
 Esterases

 Form or cleave
covalent bonds in  Aldolases
reactions other
 Decarboxylases 
Lyases than redox
 Dehydratases
reactions or
hydrolysis

 Mutases
o Move
 Converts a  Methylmalonyl-
functional
substrate into its CoA, superoxide
Isomerases groups within
isomer dismutase
a molecule
 Epimerases

Ligases
 Carboxylases
 Pyruvate carboxylase,
o Transfer
acetyl-CoA carboxylase,
 Join molecules carbon
propionyl-CoA
 Require an energy source dioxide
carboxylase
(e.g., ATP, Acetyl-CoA, groups (CO2)
 Glycogen synthase,
methylmalonyl-CoA) o Require
HMG-CoA synthase,
biotin
citrate synthase
 DNA ligase
Energy carriers
Base molecule
Transferred group Carrier of energy Released energy Metabolic site
ADP Phosphate
 Ubiquitous energy source 
ATP -31 KJ/mol
 TCA 
GDP Phosphate GTP -31 KJ/mol
Creatine Phosphate PKr
 Glycolysis
 Glycogenolysis

-43 KJ/mol  TCA
 Oxidative phosphorylation

 TCA 
CoA Thioester Acetyl-CoA -36 KJ/mol
Pyruvate Phosphate PEP
 Glycolysis 
-62 KJ/mol

Cofactors
Cofactor Vitamin Structure Reaction
Thiamine pyrophosphate (TPP) Vitamin B1
Oxidative decarboxylation
FMN/FMNH2 Vitamin B2

Electron transfer
FAD+/FADH2 Vitamin B2

Electron transfer
NAD+
/NADH Vitamin B3

Electron transfer
NADP+
/NADPH Vitamin B3

Electron transfer
 Coenzyme A Vitamin B5
Acyl group transfer
Pyridoxal phosphate Vitamin B6
Transamination, dehydration
Biotin Vitamin B7

Carboxyl group transfer

Tetrahydrofolate Vitamin B9

Methyl group transfer

Cobalamin Vitamin B12

Alkyl group transfer

S-Adenosylmethionine (SAM)
Methyl group transfer
Lipoamide
Oxidative decarboxylation
Ascorbic acid Vitamin C
Electron transfer and hydroxylation
Phylloquinone Vitamin K
Electron and carboxyl group transfer
Tetrahydrobiopterin
Electron and oxygen atom transfer
ATP
Phosphate group transfer
For more information, see vitamins.

Rate-limiting enzymes
Pathway Enzyme Stimulation Inhibition
 Citrate
 Fructose-2,6-
 Phosphofructokinase-1  ATP
biphosphate
Glycolysis (PFK-1)  Low
 AMP
pH

 Fructose-1,6-biphosphatase
Gluconeogenesis
 Fructose-2,6-
 Citrate biphosphate
 AMP
  NADH +
 Isocitrate  ADP
Citric acid H+
dehydrogenase  Ca2+
cycle  ATP

 Dephosphorylation upon
 Glycogen synthase insulin signaling
Glycogenesis
 Glucose-6-phosphate

 Phosphorylation upon
glucagon and epinephrine
signaling Glycogenolysis

 Phosphorylation upon
 Glycogen glucagon and epinephrine
Glycogenolysis phosphorylase signaling
 AMP

 Dephosphorylation upon insulin signaling

 ATP
 Glucose-6-phosphate

Pentose phosphate  Glucose-6-phosphate

 NADP+
 NADPH
pathway dehydrogenase
(HMP shunt)
 Carbamoyl phosphate
Pyrimidine synthesis synthetase II

 Phosphoribosy
l
pyrophosphate  UTP
(PRPP)
 ATP

 Glutamine-  Phosphoribosy  ADP,

phosphoribosylpyrophosphat l ATP
Purine synthesis e pyrophosphate  GDP,
(PRPP) amidotransferase (PRPP) GTP

 N-
 Carbamoyl phosphate
acetylglutamat  –
Urea cycle synthetase I
e

Fatty acid synthesis  Acetyl-CoA carboxylase

 (ACC)

 Citrate
 Phosphorylation by AMP-dependent  Acyl-CoA, e.g., palmitoyl-CoA
kinase upon ↑ AMP  Phosphorylation upon glucagon,
 Dephosphorylation upon insulin epinephrine and norepinephrine signaling
signaling

 Carnitine acyltransferase I
β-oxidation
 Induction of
expression by
 Malonyl-
long-chain fatty
CoA
acids and thyroid
hormones

 HMG-  Dephosphorylation
CoA upon insulin and thyroid  Cholesterol
Cholesterol synthesis
reductase hormone signaling

 Phosphorylation by AMP-dependent kinase upon ↑ AMP

 Phosphorylation upon glucagon signaling

Enzyme kinetics
Michaelis-Menten kinetics

 [E] = enzyme, [S] = substrate, [P] = product, [V] = velocity

o E + S ⇄ ES → E + P
 Maximum velocity (Vmax): maximum rate at which an enzyme can catalyze a reaction

Michaelis constant: (Km): the substrate concentration at which half of the active sites of the
enzymes are bound to the substrate

 Reaction velocity is ½ of Vmax when the Michaelis constant concentration is reached

 Inversely related to the affinity of the enzyme for the substrate

 Michaelis-Menten equation: v = Vmax [S] / (Km + [S])

 Km Vmax

 Directly proportional to the enzyme

 Inversely proportional to the affinity
of the enzyme for the substrate
o ↑ Enzyme affinity → ↓ Km
concentration
o ↑ Enzyme concentration → ↑ Vmax
 The only way to ↑ Vmax is to increase [E]
o Cells achieve this, e.g., by increasing
gene expression of a given enzyme
 Noncompetitive inhibitors → ↓ [E] → ↓ Vmax

Lineweaver-Burk equation and plot

The Lineweaver-Burk equation is a double reciprocal of the Michaelis-Menten equation, where

V = Vmax [S] / Km + [S] (if [E] remains constant), becomes 1 / v = Km / Vmax× 1/[S] + 1 / Vmax. It
represents enzyme kinetics in a linear graph rather than a hyperbola. This equation is particularly
important to determine the effect of drugs on enzymes.

 Intercept with y-axis: 1/Vmax: the further from zero, the lower Vmax
 Intercept with x-axis: 1/-Km : the closer to zero, the lower the affinity
 Slope: Km/Vmax

“Very efficient and kompetent”: On the Lineweaver-Burk plot, Vmax usually represents the
efficacy of a drug on the y-axis and Km represents the potency on the x-axis.

Drug-response dynamics

The details of pharmacodynamics are explained in the article on the fundamentals of

pharmacology.
Parameter Uncompetitive inhibitors
Noncompetitive inhibitors
Competitive inhibitors (reversible)
Competitive
inhibitors
(irreversible)
Similar to the  No  No  Yes  Yes
substrate
 Competitive
inhibitors
(irreversible)
 Inhibition
Effect of  None  None can be  None
increased [S] overcome

 Binds to  Do not bind to

enzyme- active site
Binding site substrate (allosteric
complex regulation)

 Binds to both
the enzyme
and enzyme-  Active site
substrate  Active site (bound
complex irreversibly)
 Reversible or
irreversible

 Decreased  None  Increased  None

Effect on Km
 Decreased  Decreased  None  Decreased
Effect on Vmax
 Does not
affect  ↓ Enzyme
 ↓ Enzyme  ↓ Enzyme
Pharmacodynamic enzyme efficacy
efficacy efficacy
effect efficacy  ↓ Potency
 ↓ Potency

For more information on the effects of inhibitors and pharmacodynamics, see types of drug-
receptor interactions.
Uncompetitive inhibitors are enzyme inhibitors that bind to the enzyme-substrate complex,
decreasing Km and Vmax.
Kompetitive Inhibition: Km Increases; Vmax unchanged. Nonkompetitive Inhibition: No Km
change; Vmax decreased.

Clinical significance
 Glucose-6-phosphate dehydrogenase deficiency
 Chronic granulomatous disease
 Lesch-Nyhan syndrome
 Von Gierke's disease
 Pompe's disease
 Cori's disease
 Andersen's disease
 McArdle's disease
 Galactosemia
 Hereditary fructose intolerance
 Essential fructosuria
 Medium chain Acyl-CoA dehydrogenase deficiency
 Mucopolysaccharidoses
 Gaucher disease
 Krabbe disease
 Tay-Sachs disease
 Fabry disease
 Metachromatic leukodystrophy
 Niemann-Pick disease
 α1-antitrypsin deficiency
 Mitochondrial myopathies
 Alkaptonuria
 Homocystinuria
 Phenylketonuria
 Histidinemia