BioChem Exam #2 Flashcards
What are the diseased states associated changes in the amounts of Hb SUBUNITS produced?
Abnormal Hb;
Alpha-thalassemia or Beta-Thalassemia;
Resulting in anemia due to decreased ability of RBC’s to transport O2 around the body
What disease results from changes in the Amino Acid sequence of Hb?
Sickle cell anemia;
Glu is replaced with Val (Non-conservative)
Changing structure changes the function!
What is the structure of normal Hb?
The OXY form has O2 bound to all subunits;
The DEOXY form exposes hydrophobic amino acids “greasy spot”on the alpha subunits, but they are inward (Glu)
What is the structure of Sickle Cell Hb?
The non-polar valine is exposed on the beta subunits and is unhappy because it is hydrophobic, so it wants to hide in the empty spots on the alpha subunits of other Hb;
Results in long chains of Hb;
These change the osmotic pressure and push water out of the RBC
What is Sickle Cell Crisis?
The collapsed cells clog up the arteries because they have formed aggregate chains;
More likely to occur in the “deoxy” state when O2 is missing because that is when the chains form;
Exercise, under anesthesia, anytime O2 is released from Hb
How does Sickle Cell help against malaria?
Malaria needs time to replicate in the body and as the spleen removes the “sick,” misshapen cells there is less time for the parasites to grow
How are proteins classified by FUNCTION?
- Structural (collagen in connective tissue)
- Transport (Hb moves Os)
- Storage (Mb stores O2)
- Defensive (Antibodies and immunity)
- Catalytic (Enzymes)
- Contractile (Actin/Myosin in muscles)
- Receptor (Hormones and communication)
- Regulatory (gene expression on/off)
- Messenger (Hormones; carry all over body)
How are proteins classified by SHAPE?
Globular;
Fibrous
What are Globular Proteins?
Long polypeptide chain folded back on itself;
Usually DON’T have repeating sequences;
ENZYMES
What are Fibrous Proteins?
Usually do contain repeating sequences;
ONE type of secondary structure;
More organized and elongated
How are proteins classified by CHEMICAL COMPOSITION?
All proteins are composed of amino acids;
Some have additional components needed for biological activity (Non-amino acid);
EX: Hb needs heme group with iron
What is a CONJUGATED PROTEIN?
Amino Acid (Apoprotein) + Non-Amino Acid (Prosthetic group)
What are some common prosthetic groups?
Metal ion (creates metalloprotein)
Carb (creates glycoprotein)
Vitamins - Riboflavin (creates flavoprotein)
What are Enzymes?
Biological catalysts of metabolic processes;
All enzymes are globular, except ribozymes (RNA);
They change the structure of the substrate and so the function! – Lower the Activation Energy
Why is Catalysis by Enzymes so important?
Without catalysis, metabolic process would proceed too slow to support life
How do Enzymes catalyze reactions?
Increase the RATE;
Increase SPECIFICITY;
Increase CONTROL
How do Enzymes increase the RATE of the reaction?
The LOWER the activation energy needed to to reach the transition state of the reaction
How do Enzymes increase the SPECIFICITY of the reaction?
Reduce the amount of waste produced by bodily reactions;
They bind ONLY certain substrates as needed
How do Enzymes increase CONTROL of the reaction?
They can change shape and therefore control enzymatic reactions and pathways;
Some have the ability to alter themselves as needed by the body
Enzymes catalyze WITHOUT…
- Altering the conditions within the cell (temp, pressure, pH, etc) - altering would denature the proteins;
- Being consumed or irreversibly altered themselves (continually recycled)
What are Substrates?
Reactant of an enzymatic reaction;
What the enzyme binds to and uses to create the product
What is Standard Free Energy change (Delta G)?
Final - Initial Energies;
(Products - Reactants);
*This product is basically fixed
Why is Standard Free Energy basically a fixed amount?
Because the substrates have one energy content and the products have another energy content no matter what;
The enzymatic difference is in how much energy it takes to TRANSFORM substrate to product
What is the Activation Energy?
Energy required to start a reaction;
Determines the rate of the reaction;
HIGH for UNcatalyzed;
LOWER for catalyzed
What is the Transition State?
Max on the curve between reactants and products;
Where there is necessary energy and atom arrangement to proceed to the products;
LOWER with ENZYME
What is Spontaneity of a Reaction?
The change in free energy (delta G);
Spontaneous, Exergonic - RELEASES energy (lower products);
Nonsponatenous, Endergonic - REQUIRES energy (higher products)
What is the Rate of a Reaction?
Amount of energy needed to reach the final product and how fast it gets there
What is the Transition State Species?
The enzyme-substrate complex;
They are bound together
How do Enzymes lower the activation energy?
- Binding reactants in close proximity (readily react b/c they are close together);
- Holding the reactants in the proper orientation (align the bonds)
- Providing the right chemical environment and correct conditions to proceed
What is the Active SIte?
Area on the enzyme, usually a cleft/crevice in the protein and certain amino acids where the substrate BINDS by non-covalent interactions and CATALYSIS occurs
What is the First Step in Catalysis?
Bind the substrate to the enzyme with highly specific interactions between the substrate and side-chains and backbone groups of the amino acids of the active site
What is the Lock-and-Key Model?
HIgh degree of similarity between the shape of the substrate and the geometry of the enzyme biding site; Complementary shapes (puzzle pieces); Does not account for protein conformational flexibility
What is the Induced-Fit Model?
Bind the substrate induces a conformational change in the enzyme causing a complementary fit after binding;
Binding site has a different 3-D shape before substrate binds;
Does account for protein conformational flexibility
What are the Steps in Enzymatic product formation?
Enzyme and Substate Separate; Enzyme + Substrate Complex; Enzyme + Product Complex; Enzyme and Product separates; Enzyme Recycled and starts over
How can the Active Site be specific?
Absolute;
Group;
Linkage;
Stereospecificity
Absolute specificity
1 enzyme, 1 substrate = 1 product
Group specificity
Substrates have a common functional group;
Enzyme looks for that one common characteristic
Linkage specificity
Substates have a common type of chemical bond
Stereospecificity
Binding due to the match up of specific arrangement of the molecules
What is the role of the R-group at the active site?
Maintain overall structure of the protein (tertiary and quaternary);
Directly participate at the active site by binding and catalyzing;
*Very responsible for the success of the catalysis
What is an Enzyme Cofactor?
Additional help for the active site;
Apozenzyme (aa) + Cofactor (Non-aa) = Holoenzyme (active)
-Prosthetic groups or
-Coenzymes
What are Prosthetic groups?
Tightly bound cofactors that are permanently apart of the of the active site
What are Coenzymes?
Loosely bound cofactors;
Come in and out of the active site as needed;
Used over and over again with the enzyme;
Largely derived from Vitamins
What is an Enzyme “assay”?
Measuring the activity of an enzyme;
How much is present
How can you measure an enzymes assay?
Measure the rate of DISAPPEARANCE of the Sub;
Measure the rate of the APPEARANCE of the Product
What type of curve is an enzyme energy profile?
Hyperbolic;
Linear - activity taking place at a constant rate;
Leveling off - all enzymes is combined to a substrate (equilibrium between formation and breakdown of E+P complexes and recycling)
Why do we only measure the INITAL rate of enzymes?
Only linear!!;
Not necessary to consider the reverse reaction;
Not necessary to consider change int eh substrate concentration;
Not necessary to reach equilibrium (then we cannot view the enzyme activity)
What are the units for expressing enzyme activity?
Rate of Reaction is used to quantitate enzymes (how much sub. is being converted to products);
International Units
How can Enzymes be used to diagnose disease?
- Direct measurement of the Enzyme concentration in the blood (The presence of Plasma specific vs. non-plasma specific enzymes) OR
- Detection/Measurement of the concentration of the substrate
What are Non-Plasma Specific Enzymes?
Present in the blood due to damaged or destroyed cells
What can be diseases are found by DIRECTLY measuring the enzyme concentration?
- Detect disease or organ damage (heart enzymes in blood after a heart attack determine damage)
- Monitoring Drug Effects (see if drug is damaging and causing release of non-specific enzymes)
- Detect predisposition to genetic diseases
What disease is diagnosed by the concentration of the Substrate? (Indirect enzyme measurement)
The measurement of the concentration of glucose in the blood
How are Enzymes used to treat disease?
Directly by injection;
Aiding in drug design
What enzymes treat by direct injection?
Streptokinase and tPA (breakdown blood clots; heart attack and stroke);
Asparaginase (destroys asparganine and limits tumor growth)
Whats problems are associated with injecting enzyme?
- Easily degraded;
- Allergic reactions (proteins);
- Cannot be absorbed into the bloodstream (not digested) so must be injected - not readily available to patients
How do enzymes help drug design?
They allow for the replication of specific inhibitors;
Targeting individual rates of metabolism
What is a biochemical pathway?
A series of linked reactions;
Enzymes causing the formation of one product which becomes the reactant of the following reaction and so on
What regulation of biochemical pathways is required?
- Maintaining order of the reactions;
- Responding to changes in the environment;
- Conserving and directing energy use to make them efficient
How are pathways regulated?
Control of the Enzyme activity that drives the rxn:
- Change the ACTIVITY level of the enzyme present (change structure)
- Change the AMOUNT of enzyme present
What are the Factors that influence Enzyme activity?
- Temperature
- pH
- Enzyme concentration
- Substrate concentration
- Presence of inhibitors
- Allosteric
- Feedback inhibition
- Regulation of Enzyme synthesis
- Location within the cell
- Isozymes
- Covalent modification
- Zymogens
How does TEMP affect enzyme activity?
Raising the temp increases the available energy to a point and reaching transition faster;
But too much heat denatures the enzymes and slows/stops the reaction
How does pH affect enzyme activity?
- Overall enzyme structure (denatures);
- Affecting the charged R-groups at the active site (altering substrate binding and catalysis);
- Change the charge on the substrate and how it interacts with the R-groups at the active site (can’t bind right)
How does Increasing Enzyme concentration effect the activity?
[Sub] remains constant, [E] increases;
Enzyme doesn’t work as hard as it can;
When all substrate is bound to reactants, can no longer see activity because no more substrate to bind;
No sub, No rxn, so enzyme can’t change anymore even though there is plenty (rain gauge)
What does the graph look like when the [S] is CONSTANT and ENZYME is INCREASING?
Y = Initial velocity; X = [E]; Hyperbolic; Levels off as the Sub becomes limiting; Only linear part can be used to determine enzyme concentration and activity
How is the concentration of the Enzyme dependent upon the substrate?
As the substrate is limited, No more conversion of enzyme;
CANNOT determine activity because it is measured by the conversion of substrate to product
How does Increasing Substrate concentration effect the enzyme activity?
Enzyme must work as hard as it can to keep up with the increasing substrate;
Enzyme eventually become limiting because all of it is bound to a substrate;
Every time a product is released, binds another substrate
What does the graph look when the [E] is CONSTANT and the [S] is INCREASING?
Y = initial velocity;
X = [S];
Hyperbolic;
Levels off as the Enzyme becomes limiting (all bound in ES);
Vmax occurs as the asymptote to enzyme concentration
*Michaelis-Menten!
What is Vmax?
the maximum reaction rate;
How fast the enzyme can convert to products;
# of substrate molecules turning to products per unit of time for a fully saturated enzyme
What is the Saturation Point for an enzyme?
When all enzymes are apart of the enzyme-substrate complex;
Steady state situation
What is the Michaelis-Menten equation?
Expresses the effect of [S] on the initial velocity;
Initial velocity = (Vmax [S]) / (Km + [S])