CHAPTER 7 FMN

ENZYMES: MECHANISM FAD

OF ACTION Thiamin pyrophosphate
biotin
ENZYMES
Catalyze chemical reaction
Act as a catalyst
are neither consumed nor permanently altered as a
consequence of their participation in a reaction METAL IONS
highly efficient constitute the most common type of prosthetic groups
extremely selective Metal ions that participate in redox reactions generally
specific not simply for the type of reaction are complexed to prosthetic groups such as heme or
catalyzed, but also for a single substrate or a small iron-sulfur clusters.
set of closely related substrates may facilitate the binding and orientation of substrates,
are also stereospecific catalysts the formation of covalent bonds with reaction
catalyze reactions of only one stereoisomer of a intermediates by acting as:
given compound Lewis acids or bases to render substrates more
For Example: d- but not l-sugars, l- but not d-amino electrophilic (electron-poor) or nucleophilic
acids (electron-rich), and hence more reactive.
they bind substrates through at least three points of
attachment, METALLOENZYMES
also can produce chiral products from nonchiral enzymes that contain tightly bound Fe, Co, Cu, Mg, Mn,
substrates and Zn

PROTEASES AND AMYLASES COFACTORS

augment the capacity of detergents to remove dirt and associate reversibly with enzymes or substrates
stains can associate either directly with the enzyme or in
the form of a cofactor-substrate complex
RENIN serve functions similar to those of prosthetic groups
utilized in the production of cheeses they bind in a transient, dissociable manner
must be present in the medium surrounding the enzyme
LACTASE for catalysis to occur
employed to remove lactose from milk for the benefit of Metal ionsmost common cofactor
lactose-intolerant persons deficient in this hydrolytic
enzyme METAL ACTIVATED ENZYMES
Enzymes that require metal ion cofactor
ASE
a descriptor for the type of reaction catalyzed COENZYMES
serves as a substrate shuttle
DEHYDROGENASES serve as recyclable shuttles that transport many
enzymes that remove hydrogen atoms substrates from one point within the cell to another
Functions of shuttle:
PROTEASES Stabilize species such as hydrogen atoms (FADH) or
enzymes that hydrolyze proteins hydride ions (NADH) that are too reactive to persist
for any significant time in the presence of the water
SIX CLASSES OF ENZYMES or organic molecules that permeate cells.
Oxidoreductases serve as an adaptor or handle
enzymes that catalyze oxidations and reductions facilitates the recognition and binding of small
Transferases chemical groups, such as acetate (coenzyme A)
enzymes that catalyze transfer of moieties such as or glucose (UDP), by their target enzymes
glycosyl, methyl, or phosphoryl groups Other chemical moieties transported by coenzymes
Hydrolases include:
enzymes that catalyze hydrolytic cleavage of CC, methyl groups (folates)
CO, C N, and other covalent bonds oligosaccharides (dolichol)
Lyases
enzymes that catalyze cleavage of CC, CO, MANY COENZYMES, COFACTORS, & PROSTHETIC
CN, and other covalent bonds by atom GROUPS ARE DERIVATIVES OF B VITAMINS
elimination, generating double bonds Nicotinamide
Isomerases is a component of the redox coenzymes NAD and
enzymes that catalyze geometric or structural NADP
changes within a molecule Riboflavin
Ligases is a component of the redox coenzymes FMN and
enzymes that catalyze the joining together (ligation) FAD
of two molecules in reactions coupled to the Pantothenic acid
hydrolysis of ATP is a component of the acyl group carrier coenzyme A
Thiamin
PLAY IMPORTANT ROLES IN CATALYSIS Participates in decarboxylation of a-keto acids
prosthetic groups Folic acid and cobamide coenzymes
cofactors function in one-carbon metabolism
coenzymes
CATALYSIS
PROSTHETIC GROUPS occurs at active site
are tightly integrated into an enzymes (protein) is further enhanced by the capacity of the active site to
structure by covalent or noncovalent forces shield substrates from water and generate an
Ex: environment whose polarity, hydrophobicity, acidity, or
 alkalinity can differ markedly from that of
surrounding cytoplasm
EMIL FISCHER
propose that enzymes and their substrates interact to
form an enzyme-substrate (ES) complex whose thermal
stability was greater than that of the enzyme itself
He reasoned that the exquisitely high specificity with
which enzymes discriminate their substrates when
forming an ES complex was analogous to the manner in
which a mechanical lock distinguishes the proper key.
lock and key model
CATALYSIS BY PROXIMITY
For molecules to interact, they must come within bond-
forming distance of one another.
The higher their concentration:
the more frequently they will encounter one
another, and the greater will be the rate of their
reaction
ACID-BASE CATALYSIS
the ionizable functional groups of aminoacyl side chains,
and where present of prosthetic groups, can contribute
to catalysis by acting as acids or bases
2 Types:
Specific acid or base catalysis
refers to reactions for which the only
participating acid or base are protons or
hydroxide ions
The rate of reaction is:
sensitive to changes inthe concentration of
protons or hydroxide ions, but
independent of the concentrations of other
acids (proton donors) or bases (proton
acceptors) present in the solution or at the
active site
General acid catalysis or general base catalysis
Reactions whose rates are responsive to all the
acids or bases present.
CATALYSIS BY STRAIN
Enzymes that catalyze lytic reactions, chemical
transformations that involve breaking a covalent bond,
typically bind their substrates in a conformation that is
somewhat unfavorable for the bond targeted for
cleavage.
this strained conformation mimics that of the
transition state intermediate
Transition state intermediate
a transient species that represents the transition
state, or midway point, in the transformation of
substrates to products
The resulting strain selectively stretches or distorts the
targeted bond, weakening it and making it more
vulnerable to cleavage.
LAUREATE LINUS PAULING
was the first to suggest a role for transition state
stabilization as a general mechanism by which enzymes
accelerate the rates of chemical reactions
the is particularly common among enzymes that catalyze
group transfer reactions
Residues on the enzyme that participate in covalent
catalysis generally are:
cysteine or serine
occasionally histidine
often follows a ping-pong mechanism
one in which the first substrate is bound and its
product released prior to the binding of the second
substrate
SUBSTRATES
induce conformational changes in enzymes
INDUCE FIT MODEL
proposed by Daniel Koshland
states that when a substrate approach and bind to an
enzyme they induce a conformational change that is
analogous to placing a hand (substrate) into a glove
(enzyme).
The enzyme in turn induces reciprocal changes in its
substrate harnessing the energy of binding to
facilitate the transformation of substrate into
products.
HIV PROTEASE
illustrates acid-base catalysis
Enzymes of the aspartic protease family, which includes:
the digestive enzyme pepsin
the lysosomal cathepsins
the protease produced by the human
immunodeficiency virus (HIV)
They share a common mechanism that employs two
conserved aspartyl residues as acid-base catalysts.
In the first stage of the reaction, an aspartate
functioning as a general base, extracts a proton
from a water molecule, making it more nucleophilic.
The resulting nucleophile then attacks the
electrophilic carbonyl carbon of the peptide
bond targeted for hydrolysis, forming a
tetrahedral transition state intermediate.
A second aspartate then facilitates the
decomposition of this tetrahedral intermediate by
donating a proton to the amino group produced by
rupture of the peptide bond.
The two active site aspartates can act simultaneously as
a general base or as a general acid because their
immediate environment favors ionization of one, but not
the other.
Catalysis by aspartic proteases
involves the direct hydrolytic attack of water on a
peptide bond

COVALENT CATALYSIS
involves the formation of a covalent bond between the
enzyme and one or more substrates
The modified enzyme thus becomes a reactant.
introduces a new reaction pathway whose activation
energy is lowerand the reaction therefore is faster
than the reaction pathway in homogeneous solution
The chemically modified state of the enzyme is,
however, transient.
Completion of the reaction returns the enzyme to its ILLUSTRATE COVALENT CATALYSIS
original, unmodified state. Its role thus remains catalytic. Chymotrypsin
Fructose-2, 6-bisphosphatase
 the simultaneous synthesis of large libraries of chemical
CHYMOTRYPSIN compounds that contain all possible combinations of a
catalysis by the serine protease chymotrypsin set of chemical precursors
involves formation of a covalent acylenzyme Enzyme assays that produce a chromogenic or
intermediate fluorescent product are ideal, since optical detectors are
Serine 195 readily engineered to permit the rapid analysis of
A conserved seryl residue multiple samples, often in real time.
Is activated via interactions with histidine 57 and Principal use:
aspartate 102. analysis of inhibitory compounds with ultimate
Charge-relay network potential for use as drugs
Linked formed by Asp 102-His 57-Ser 195 (in order)
ENZYME-LINKED IMMUNOASSAYS (ELISAs)
Act as proton shuttle
use antibodies covalently linked to a reporter enzyme
such as:
alkaline phosphatase or horseradish peroxidase
whose products are readily detected, generally
by the absorbance of light or by fluorescence
STEPS:
Serum or other biologic samples to be tested are placed
in plastic, multi-well microtiter plates, where the proteins
adhere to the plastic surface and are immobilized.
Any exposed plastic that remains is subsequently
blocked by adding a nonantigenic protein such as
bovine serum albumin.
A solution of antibody covalently linked to a reporter
enzyme is then added.
The antibodies adhere to the immobilized antigen
and are themselves immobilized.
Excess free antibody molecules are then removed by
washing.
FRUCTOSE-2,6-BISPHOSPHATASE The presence and quantity of bound antibody is then
a regulatory enzyme of gluconeogenesis determined by adding the substrate for the reporter
catalyzes the hydrolytic release of the phosphate on enzyme.
carbon 2 of fructose-2,6-bisphosphate
Catalysis involves a NAD(P)+-DEPENDENT DEHYDROGENASES
catalytic triad are assayed spectrophotometrically
one Glu
two His residues
a covalent phosphohistidyl intermediate

ISOZYMES
are distinct enzyme forms that catalyze the same
reaction
protein catalyst
arise from gene duplication
Isozymes that catalyze the identical reaction may also
enhance survival by providing a backup copy of an
essential enzyme.
SINGLE-MOLECULE ENZYMOLOGY
Is a process that measure the rate of single catalytic
events and sometimes the individual steps in catalysis
by a process
HIGH-THROUGHPUT SCREENING
takes advantage of robotics, optics, data processing, and
microfluidics to conduct and analyze many thousands of
assays of the activity of a given enzyme simultaneously
is ideal for surveying the numerous products of
combinatorial chemistry
SPECTROPHOTOMETRIC ASSAYS
exploit the ability of a substrate or product to absorb
light
absorb light at a wavelength of 340 nm:
reduced coenzymes NADH and NADPH, written as
NAD(P)H,
whereas their oxidized forms NAD(P)+ do not
When NAD(P)+ is reduced:
the absorbance at 340 nm therefore increases in
proportion toand at a rate determined bythe
quantity of NAD(P)H produced
Conversely, for a dehydrogenase that catalyzes the
oxidation of NAD(P)H:
a decrease in absorbance at 340 nm will be
observed
The rate of change in absorbance at 340 nm will be
proportionate to the quantity of the enzyme present.
For example, hydrolysis of the phosphoester bond in p-
nitrophenyl phosphate (pNPP):
an artificial substrate molecule, is catalyzed at a
measurable rate by numerous phosphatases,
phosphodiesterases, and serine proteases
While pNPP does not absorb visible light, following
its hydrolysis the resulting p-nitrophenylate anion
absorbs light at 419 nm, and thus can be quantified.
BIOMARKERS
molecules whose appearance or levels can assist in the
diagnosis and prognosis of diseases and injuries
affecting specific tissues

FIRST ENZYMES USED TO DIAGNOSE MI
aspartate aminotransferase (AST)
alanine aminotransferase (ALT)
lactate dehydrogenase (LDH)
AST & ALT
proved less than ideal
appear in plasma relatively slowly
not specific to heart muscle
LACTATE DEHYDROGENASE (LDH)
is released relatively slowly into plasma
it offered the advantage of tissue specificity as a
consequence of its quaternary structure
is a tetrameric enzyme consisting of two monomer
types:
H (for heart)
M (for muscle)
combine to yield five LDH isozymes:
HHHH (I1) predominates in heart tissue
HHHM (I2)
HHMM (I3)
HMMM (I4)
MMMM (I5) predominates in liver
When LDH levels rise in blood plasma:
the identity of the injured tissue can be inferred
from its characteristic pattern of LDH isozymes
can be separated by electrophoresis and detected using
a coupled assay
CREATINE KINASE (CK)
has three isozymes:
CK-MM (skeletal muscle)
CK-BB (brain)
CK-MB (heart and skeletal muscle)
has a useful diagnostic window
It appears within: 4 to 6 hours of an MI
peaks at: 24 hours
returns to a baseline level by: 48 to 72 hours
individual CK isozymes are separable by electrophoresis,
thus facilitating detection
TROPONINS
is a complex of three proteins involved in muscle
contraction in skeletal and cardiac muscle but not in
smooth muscle
Immunological measurement of plasma levels of cardiac
troponins I and T provide sensitive and specific
indicators of damage to heart muscle.
Troponin levels
rise for 2 to 6 hours after an MI, remain elevated for
4 to 10 days
In addition to MI, other heart muscle damage also
elevates serum troponin levels.
Cardiac troponins
Serve as a marker of all heart muscle damage
CLINICAL USES OF ENZYMES
Glucose oxidase
frequently utilized to measure plasma glucose
concentration
Tissue plasminogen activator (tPA) or streptokinase
is used in the treatment of acute MI
Trypsin
has been used in the treatment of cystic fibrosis
Intravenous infusion of recombinantly produced
glycosylases has been approved for the treatment of
several lysosomal storage diseases including the:
Gaucher disease -glucosidase
Pompe disease -glucosidase
Fabry disease -galactosidase A
Sly disease -glucuronidase
RESTRICTION ENDONUCLEASES
Enzymes that cleave double-stranded DNA at sites
specified by a sequence of four, six, or more base pairs
called restriction sites.
Cleavage of a sample of DNA with a restriction enzyme
produces a characteristic set of smaller DNA fragments.
RESTRICTION FRAGMENT LENGTH POLYMORPHISMS
(RFLPS)
deviation in the normal product pattern
occur if a mutation renders a restriction site
unrecognizable to its cognate restriction endonuclease
or, alternatively, generates a new recognition site
are currently utilized to facilitate prenatal detection of a
number of hereditary disorders, including sickle cell trait,
-thalassemia, infant phenylketonuria, and Huntington
disease
POLYMERASE CHAIN REACTION (PCR)
employs a thermostable DNA polymerase and
appropriate oligonucleotide primers to produce
thousands of copies of a defined segment of DNA from a
minute quantity of starting material
enables medical, biological, and forensic scientists to
detect and characterize DNA present initially at levels
too low for direct detection
used to detect and identify pathogens and parasites
such as:
Trypanosoma cruzi
the causative agent of Chagas disease
Neisseria meningitides
the causative agent of bacterial meningitis
RECOMBINANT FUSION PROTEINS
are purified by affinity chromatography
The gene of interest is linked to an oligonucleotide
sequence that encodes a carboxyl or amino terminal
extension to the encoded protein.
The resulting modified protein, termed a fusion
protein, contains a new domain tailored to interact
with an appropriately modified affinity support
SITE-DIRECTED MUTAGENESIS
facilitates identification of the specific roles of given
aminoacyl residues in substrate binding and catalysis
used to change residues suspected of being important in
catalysis or substrate binding
provides insights into mechanisms of enzyme action
THOMAS CECH
discovered the first catalytic RNA molecule
 Many enzymes can be assayed spectrophotometrically
NOTE by coupling them to an NAD(P)+-dependent
Not all enzymes are proteins. dehydrogenase.
Several ribozymes are known that can cut and re-splice
the phosphodiester bonds of RNA.
In the ribosome, it is the rRNA are primarily responsible
for catalysis.
Aminoacyl residues that participate in catalysis are
highly conserved among all classes of a given enzyme.
The catalytic activity of enzymes reveals their presence,
facilitates their detection, and provides the basis for
enzyme-linked immunoassays.