Enzymes

5/13/2021
Enzymes
Chapter 8
Copyright © 2017 by Elsevier, Inc. All rights reserved.
Introduction to Enzymes
 Biological catalysts
➢ Neither consumed nor permanently altered
 Enzyme concentrations
➢ Detect cellular injury
➢ Identify abnormalities that cause disease
Copyright © 2017 by Elsevier, Inc. All rights reserved. 2
Enzyme Structure
 Enzyme structure and properties

➢ Primary structure
➢ Secondary structure
➢ Tertiary structure
➢ Quaternary structure
 Enzyme naming
1
5/13/2021
Enzyme Activity
 Very efficient
 Substrate: what it uses to create product
 Active site: where substrate binds by
noncovalent bond
 Enzyme substrate complex
 Allosteric site: binding of regulator molecules
(can change the shape of the enzyme)
 Enzyme locations
Reaction Rates of Chemical

Reactions
 Enzyme kinetics:
➢ rate of chemical reaction
➢ can change over the course of rxn
 Product formation
➢ Affected by enzyme concentration
➢ Proportional to enz. concentration as long as there
is substrate excess
 Reaction rate factors: pH, temp, cofactors,
inhibitors
 Assessing enzyme activity: linear phase
Reaction rate curves with 3 different

amounts of enzyme.
1 has low-enzyme activity,
2 has medium-enzyme activity,
3 has high-enzyme activity.
Increased enzyme activity results in
decreased lag phase, decreased linear
phase, and a plateau phase of substrate
depletion that is reached faster.
2
5/13/2021
Thermodynamics
 Transition state: energy barrier to be overcome

 Activation energy: needed energy for
➢ Enzymes lower activation energy
 Two-step reaction
Michaelis-Menten Model
 Based on concentration of enzyme and
substrate
Vmax [S]
V0 =
Km + [S]
➢ V0 is the initial velocity (product concentration is
zero). S is the substrate concentration. Both V max
and Km are constants that characterize specific
enzymes
 Increased reaction rate until substrate binding
sites are depleted
3
5/13/2021
Enzyme Inhibition
 Competitive
➢ competitor binds to the active binding site on the
enzyme, prevents substrate from binding
➢ reversible
 Noncompetitive
➢ competitor binds to allosteric site
➢ irreversible
 Uncompetitive
➢ Binds the enzyme substrate complex
Activators, Coenzymes, and

Prosthetic Groups
 Activators—molecules that bind and increase
activity
➢ Cofactors: magnesium and calcium
 Coenzymes—organic substances loosely
bound,
➢ help the rxn; ex - NAD
 Prosthetic groups—tightly bound molecules
Enzyme Reaction Conditions
 pH and temperature
 Enzyme concentration
➢ Zero order kinetics
 Substrate concentration
➢ First order kinetics
 Presence of activators
 Presence of inhibitors
4
5/13/2021
Isoenzymes
 Catalyze same reaction, different structural

and biochemical properties
 Commonly measured isoenzymes
➢ CK-MB
 Laboratory techniques
➢ Electrophoresis
➢ Immunoassay
Enzyme Measurements
 Performed on serum, plasma, body fluids
 Very low concentrations
 End point assays
➢ Fixed time period, amount of product is measured
 Kinetic assays
➢ Reaction monitored continuously over time
➢ Most assays
➢ Needs to be read in zero order
 IU/L
➢ Measure of activity
➢ amount of enzyme required to convert 1 μmol of
substrate into product in 1 minute
14
Aminotransferases
 Physiology: catalyze the transfer of amino
groups from amino acids to form 2-oxo-acids
➢ AST: Makes L-glutamate and oxaloacetate
➢ ALT: Makes L-glutamate and pyruvate
 Clinical significance:
➢ Both found in all major organs
➢ AST is primarily found in the heart, liver, skeletal
muscle, and kidney, and is found in the cytoplasm
and the mitochondria.
➢ ALT is found in the cytoplasm only, and is
primarily seen in the liver and kidney.
5
5/13/2021
Aminotransferases
 Clinical significance:
➢ AST & ALT are part of the comprehensive
metabolic panel (CMP)
➢ AST & ALT measured when evaluating a patient
for liver disease (elevated in liver disease).
• Reflect hepatocellular destruction (viral hepatitis)
• Elevated enzyme levels, often seen before the patient
has symptoms.
• Elevations of AST and ALT can be seen as much as 100
times the reference range.
➢ ALT has greater specificity for liver disease than
does AST.
Aminotransferases
 Laboratory procedures
➢ measured using a coupled reaction to a specific
dehydrogenase reaction
➢ ultimately measuring the conversion of NAD to
NADH at 340 nm.
 Hemolysis should be avoided when
measuring AST or ALT because both are
present in large quantities in cellular
cytoplasm.
Alkaline Phosphatase
 Physiology
➢ Hydrolase releases inorganic phosphate from its
substrates
➢ found on membranes and cell surfaces of the
small intestines, kidneys, liver, bone, and
placenta.
 Clinical significance
➢ Part of CMP
➢ increases in ALP activity are commonly seen in
obstructive hepatobiliary diseases as well as in
osteoblast-mediated diseases
6
5/13/2021
Alkaline Phosphatase
➢ Reference ranges for infants and children are
higher than in adults due to elevated activity
during growth periods
➢ Pregnancy elevates ALP
➢ Hemolysis
➢ Heat inactivation
• distinguish ALP activity derived from liver or bone.
• ALP from the liver will remain after heating the sample;
that from bone will be destroyed by heat.
γ-Glutamyl Transferase
 Physiology
➢ exact function of GGT is unknown;
➢ catalyzes the transfer of a γ-glutamyl from a
peptide.
➢ found on the cell surface and in the cytoplasm.
➢ GGT found in serum is primarily of hepatic origin.
➢ Increased GGT levels are seen in a majority of
liver disorders with unusually high levels seen in
patients with primary and metastatic
hepatocellular carcinoma.
γ-Glutamyl Transferase
➢ Patients with alcoholic cirrhosis or chronic
alcoholics also display elevated GGT activity.
 Laboratory methods
➢ often measured when ALP activity is elevated
➢ If the elevated ALP activity is of hepatic origin,
both the GGT and ALP will be elevated;
➢ if elevated ALP is from bone, GGT will be normal.
➢ Hemolysis does not affect GGT levels.
7
5/13/2021
Creatine Kinase (CK)
 Physiology
➢ CK catalyzes the reversible phosphorylation of
creatine to creatine phosphate by ATP.
➢ The forward reaction occurs in the mitochondria.
• ATP -> ADP and stores energy in the form of creatine
phosphate.
➢ The reverse reaction occurs in the cytoplasm:
• ADP -> ATP. This production of energy is necessary for
muscular contraction
 Isoforms
➢ Subunits: M and B (CK-MM (98%), CK-MB, CK-BB)
Creatine Kinase (CK)

➢ Total CK elevations are seen after any kind of
muscular injury or trauma.
➢ Muscular diseases such as muscular dystrophy
and rhabdomyolysis show extreme elevations.
➢ Acute myocardial infarctions (AMI) also increase
the total CK.
➢ CK by coupled reaction; final step is the oxidation
of NADP+ to NADPH
➢ Immunoassay: CK-MB for heart attacks
Lactate Dehydrogenase (LD)
 Physiology:
➢ LD catalyzes the reversible oxidation of lactate to
pyruvate.
➢ LD activity exists in all cells with concentrations
higher in cytoplasm than in serum.
➢ Significant activity is seen in heart, liver, red blood
cells, kidney, and skeletal muscle.
➢ Isoenzymes (M and H)
➢ elevations can be seen in a variety of disorders
8
5/13/2021
Lactate Dehydrogenase (LD)
➢ Significant elevations can be seen in pernicious
anemia, megaloblastic anemia, and some
cancers.
➢ LD activity is measured using the forward
catalyzed reaction of lactate to pyruvate.
➢ LD is found in red blood cells; thus, hemolyzed
samples are unacceptable for analysis.
Amylase
 Physiology
➢ catalyzes the hydrolysis of α-glycosidic bonds in
order to release various sugars from starches
➢ from acinar cells of the pancreas and the salivary
glands
➢ greatest elevations of serum amylase activity are
seen in acute pancreatitis and salivary gland
inflammation
• to diagnose acute pancreatitis need AMY and LIP
• peaks at 12 hours and returns to normal in 3-4 days
Amylase
➢ Coupled enzyme reactions resulting in the
measurement of NAD+ at 340 nm.
➢ Testing is available to measure the activities of p-
AMY and s-AMY.
9
5/13/2021
Lipase
 Physiology:
➢ catalyzes the hydrolysis of triglycerides into
glycerol and fatty acids.
➢ Lipase exists almost exclusively in the pancreas
➢ pancreatic origin (almost exclusively)
➢ During acute pancreatitis, lipase levels peak at 24
hours and don’t return to normal until 8 to 14 days
later.
➢ Increased lipase activity is more specific than
amylase activity when diagnosing acute
pancreatitis.
28
10

Enzymes

Uploaded by

Document Information

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

Enzymes

Uploaded by

Copyright:

Available Formats

5/13/2021

Copyright © 2017 by Elsevier, Inc. All rights reserved.

Copyright © 2017 by Elsevier, Inc. All rights reserved. 2

 Enzyme structure and properties

Copyright © 2017 by Elsevier, Inc. All rights reserved. 3

Copyright © 2017 by Elsevier, Inc. All rights reserved. 4

Reaction Rates of Chemical

Reaction rate curves with 3 different

 Transition state: energy barrier to be overcome

Copyright © 2017 by Elsevier, Inc. All rights reserved. 7

Copyright © 2017 by Elsevier, Inc. All rights reserved. 9

Copyright © 2017 by Elsevier, Inc. All rights reserved. 10

Activators, Coenzymes, and

Copyright © 2017 by Elsevier, Inc. All rights reserved. 11

Enzyme Reaction Conditions

Copyright © 2017 by Elsevier, Inc. All rights reserved. 12

 Catalyze same reaction, different structural

Copyright © 2017 by Elsevier, Inc. All rights reserved. 13

Copyright © 2017 by Elsevier, Inc. All rights reserved. 16

Copyright © 2017 by Elsevier, Inc. All rights reserved. 17

Copyright © 2017 by Elsevier, Inc. All rights reserved. 21

Creatine Kinase (CK)

Creatine Kinase (CK)

Copyright © 2017 by Elsevier, Inc. All rights reserved. 23

Lactate Dehydrogenase (LD)

Copyright © 2017 by Elsevier, Inc. All rights reserved. 24

Lactate Dehydrogenase (LD)

Copyright © 2017 by Elsevier, Inc. All rights reserved. 25

Copyright © 2017 by Elsevier, Inc. All rights reserved. 27

You might also like