Uploaded by jenbubble_


Justine Arla Gustilo, RMT
Biomedical importance:
- presence and maintenance of a complete set of enzyme
is essential for breakdown of nutrients to supply energy
and chemical building blocks.
- deficiency in quantity / catalytic activity of key enzyme
can result from genetics, nutritional deficit / toxins
biological polymers that catalyze chemical reaction
are proteins
enhance the rate of corresponding non-catalyzedreaction
neither consumed nor altered as a consequence
of their participation in a reaction.
• highly efficient
• extremely selective catalyst
• 1. specific for the type of reaction catalyzed
• 2. for a single substrate or a small set of
closely related substrate.
• 3. stereospecific catalyst, catalyzing reaction
only of one stereoisomer of a given
- binds substrate through at least 3 points of attachment |
→ chiral product
- many contain small non-protein molecules and metal ions
that participate directly in substrate binding / catalysis
(prosthetic group, cofactors, and coenzyme)
Names: -ase
dehydrogenase – remove hydrogen atom
protease – hydrolyze protein
isomerase – catalyze rearrangement in configuration
6 classes of enzyme according to IUB
1. oxidoreductase
2. transferase
3. hydrolases
4. lyases
5. Isomerases
6. ligase
Prosthetic groups – tightl and stable incorporation into a
protein’s structure by covalent / noncovalent forces
ex. Pyridoxal phosphate, FMN, FAD, thiamine pyrophosphate,
biotin, metal ions ( Cu, Co, Mg, Mn, Zn, Fe)
• Metalloenzyme
Function of metal ions bound to enzyme:
1. facilitate binding and orientation of substrate
2. formation of covalent bonds with reaction intermediate
(Co2+ in coenzyme B12)
3. interact with substrate to render them more
electrophillic or nucleophillic
Metal ions in redox reaction are complexed to prosthetic groups
ex. Heme, iron-sulfur cluster
Cofactors – binds in transient, dissociable manner, either to
the enzyme or to the substrate such as ATP
- must be present in the medium surrounding the
enzyme for catalysis to occur
• Metal-activated enzyme
Coenzymes – serves as recyclable shuttles (group transfer
agents) that transport many substrate from their point of
generation to their point of utilization
- stabilizes substrate ( H atom / hydride ion) in aqueous
environment of the cell.
- transport methyl group (folate) acyl group (coenzyme A),
oligosaccharides (dolichol)
Sources: Vitamin B
¤important component of coenzyme
¤ pantothenic acid
¤ folic acid and cobamide
¤ thiamine pyrophosphate
¤ nicotinamide
¤ riboflavin
Active site
› environment that is exquisitely tailored for single reaction
› takes the form of a cleft or pocket.
› 3-dimentional & shield substrate from solvent and
facilitates catalysis
› binds and orient cofactors or prosthetic groups
4 general mechanism used to enhance catalytic rate :
1. catalysis by proximity
- enzyme binded to substrate creates a region of high local
substrate concentration
- also orient the substrate molecule spatially in a position for
them to interact
2. acid-base catalysis
act as
ionizable functional group of amino acyl side chain
prosthetic group
kinds: a). Specific
- rate of reaction is sensitive to changes of
proton but independent of the concentration of other
acid/base present in the solution or at the active site
b). General – rate of reaction is responsive to all the
acid / base present
3. Catalysis by strain
- enzyme binds their substrate in a conformation
slightly unfavorable for the bond that will undergo cleavage.
4. Covalent catalysis
- involve the formation of a covalent bond between
the enzyme and one or more substrate  modified
enzyme and becomes a reactant
- induces a new reaction pathway whose activation
energy is lower
- chemical modification in enzyme is transient and it
returns to its original form after completion of rxn
- common among enzyme that catalyze grp transfer rxn
- follows ping-pong mechanism
Emil Fischer – compared the highly specific fit between the
enzyme and its substrate to that of a lock and key.
David Koshland induced fit model – when substrate approach
and binds to an enzyme they induce a conformational change (
analogous to a hand and a glove)
› physically distinct version of a given enzyme, each
of which catalyze the same reaction.
› found in higher animals
› arises through gene duplication
› exhibit subtle difference in properties, like, sensitivity
to particular regulatory factor or substrate affinity
that adopt them to certain tissue / circumtances
› provide back up copy of an essential enzyme
Isozyme of lactate dehydrogenase:
L-Lactate dehydrogenase is a tetrameric enzyme with 4 subunits in 2
isoform ( H / M )
isozyme I1 predominantly H – heart
I5 predominantly M – liver
- rate of catalytic reaction being monitored is proportionate to the
amount of enzyme present – can infer enzyme concentration
Enzyme detection:
1. Single molecule enzymology – measures rate of single catalytic event
and for individual steps in catalysis.
- uses fluorescence microscopy
2. High-throughput screening – uses optical detectors which are
engineered to permit rapid analysis of multiple samples, thus
a). Ideal for survey of numerous product of combinational chemistry
b). Simultaneous synthesis of large libraries of chemical compound
that contain all possible combination of sets of chemical precursors.
- enzyme assay that produce a chromatogenic / fluorescent product
3. enzyme-linked immunoassay (ELISA) – detect protein that lack
catalytic activity
4. Spectrophotometric assay
-exploit the ability of a substrate / product to absorb light
Functional plasma enzyme – perform a physiologic function I
n the blood.
- lipoprotein, lipase, pseudocholinesterase, proenzyme of
blood coagulation and blood clot dissolution
- majority are synthesized and secreted by the liver
Nonfunctional plasma enzyme – enzyme in the plasma that
perform no known physiologic function in the blood
Detection of gene mutation using technique that relay on the catalytic
efficiency and specificity of enzyme catalyst
1. polymerase chain reaction (PCR) – relies upon the ability of enzyme to
serve as catalytic amplifiers to analyze the DNA present in biologic and
forensic samples
- thermostable DNA polymerase = appropriate oligonucleotide
primer → thousands of copies of a sample of DNA to be detected
2. restriction fragment length polymorhism (RFLPs) – facilitates prenatal
detection of hereditary disorder e.g., sickle cell trait, -thalassemia, PKU,
huntington’s disease
- involves cleavage of double stranded DNA by restriction
endonucleases, which can detect subtle alteration in DNA that affect their
recognized site