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Biochem 7
Biochem 7
Biochem 7
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
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
ISOZYMES observed
are distinct enzyme forms that catalyze the same The rate of change in absorbance at 340 nm will be
reaction proportionate to the quantity of the enzyme present.
protein catalyst For example, hydrolysis of the phosphoester bond in p-
arise from gene duplication nitrophenyl phosphate (pNPP):
Isozymes that catalyze the identical reaction may also an artificial substrate molecule, is catalyzed at a
enhance survival by providing a backup copy of an measurable rate by numerous phosphatases,
essential enzyme. phosphodiesterases, and serine proteases
While pNPP does not absorb visible light, following
SINGLE-MOLECULE ENZYMOLOGY its hydrolysis the resulting p-nitrophenylate anion
Is a process that measure the rate of single catalytic absorbs light at 419 nm, and thus can be quantified.
events and sometimes the individual steps in catalysis
by a process BIOMARKERS
molecules whose appearance or levels can assist in the
HIGH-THROUGHPUT SCREENING diagnosis and prognosis of diseases and injuries
takes advantage of robotics, optics, data processing, and affecting specific tissues
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
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
FIRST ENZYMES USED TO DIAGNOSE MI Enzymes that cleave double-stranded DNA at sites
aspartate aminotransferase (AST) specified by a sequence of four, six, or more base pairs
alanine aminotransferase (ALT) called restriction sites.
lactate dehydrogenase (LDH) Cleavage of a sample of DNA with a restriction enzyme
produces a characteristic set of smaller DNA fragments.
AST & ALT
proved less than ideal RESTRICTION FRAGMENT LENGTH POLYMORPHISMS
appear in plasma relatively slowly (RFLPS)
not specific to heart muscle deviation in the normal product pattern
occur if a mutation renders a restriction site
LACTATE DEHYDROGENASE (LDH) unrecognizable to its cognate restriction endonuclease
is released relatively slowly into plasma or, alternatively, generates a new recognition site
it offered the advantage of tissue specificity as a are currently utilized to facilitate prenatal detection of a
consequence of its quaternary structure number of hereditary disorders, including sickle cell trait,
is a tetrameric enzyme consisting of two monomer -thalassemia, infant phenylketonuria, and Huntington
types: disease
H (for heart)
M (for muscle) POLYMERASE CHAIN REACTION (PCR)
combine to yield five LDH isozymes: employs a thermostable DNA polymerase and
HHHH (I1) predominates in heart tissue appropriate oligonucleotide primers to produce
HHHM (I2) thousands of copies of a defined segment of DNA from a
HHMM (I3) minute quantity of starting material
HMMM (I4) enables medical, biological, and forensic scientists to
MMMM (I5) predominates in liver detect and characterize DNA present initially at levels
too low for direct detection
When LDH levels rise in blood plasma:
used to detect and identify pathogens and parasites
the identity of the injured tissue can be inferred
such as:
from its characteristic pattern of LDH isozymes
Trypanosoma cruzi
can be separated by electrophoresis and detected using
the causative agent of Chagas disease
a coupled assay
Neisseria meningitides
CREATINE KINASE (CK) the causative agent of bacterial meningitis
has three isozymes:
CK-MM (skeletal muscle) RECOMBINANT FUSION PROTEINS
are purified by affinity chromatography
CK-BB (brain)
The gene of interest is linked to an oligonucleotide
CK-MB (heart and skeletal muscle)
sequence that encodes a carboxyl or amino terminal
has a useful diagnostic window
extension to the encoded protein.
It appears within: 4 to 6 hours of an MI
The resulting modified protein, termed a fusion
peaks at: 24 hours protein, contains a new domain tailored to interact
returns to a baseline level by: 48 to 72 hours with an appropriately modified affinity support
individual CK isozymes are separable by electrophoresis,
thus facilitating detection SITE-DIRECTED MUTAGENESIS
facilitates identification of the specific roles of given
TROPONINS aminoacyl residues in substrate binding and catalysis
is a complex of three proteins involved in muscle used to change residues suspected of being important in
contraction in skeletal and cardiac muscle but not in catalysis or substrate binding
smooth muscle provides insights into mechanisms of enzyme action
Immunological measurement of plasma levels of cardiac
troponins I and T provide sensitive and specific THOMAS CECH
indicators of damage to heart muscle. 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.