Biochem Lesson 19

AMP

AMP also provides a regulatory signal to glycogen phosphorylase. It binds to the same site as ATP, but it stimulates glycogen phosphorylase rather than inhibiting it (Figure 15.15c). AMP acts as a positive effector, meaning that it enhances the binding of substrate to glycogen phosphorylase. Significant levels of AMP indicate that the energy status of the cell is low and that more energy (ATP) should be produced.

negative heterotropic effectors

ATP decreases the enzyme's activity by decreasing the affinity of the enzyme for phosphate ions. ATP and glucose-6-phosphate are therefore negative heterotropic effectors (they decrease the affinity of a different molecule, i.e. phosphate ion). This type of allosteric inhibition will increase the value of KM but not affect the VMax since the active site is unaffected.

What type of enzyme is affected by the binding of a regulating substance at a point other than the active site?

Allosteric

Glycogen phosphorylase is a dimer of two identical subunits of 831 amino acids. Each subunit has the following structural attributes:

An active site for the phosphorolysis of glycogen A bound pyridoxal phosphate cofactor A glycogen binding site An allosteric effector site near the interface between the two subunits An interface region that includes an a-helix "tower" that extends into, and packs against, the other subunit

Effect of product accumulation

As product builds up, the net forward rate of reaction may slow, as a significant rate of reverse reaction occurs (i.e. as equilibrium is reached). Also, some products may actually bind to and inhibit the enzyme. This is called product inhibition

What name is given to substances that cause loss of activity in an enzyme? A) inactivator B) inhibitor C) deactivator D) zymogen E) proenzyme

B) inhibitor

What type of allosteric control results when a regulator increases the reaction rate by making the active site bind to more substrate? A) enhanced B) negative C) positive D) noncompetitive E) subdued

C) Positive

What name is given to the inactive precursor of an enzyme? A) preenzyme B) antigen C) zymogen D) enzymogen E) enzyte

C) zymogen

Covalent modification of the enzyme molecule

Covalent modification of an enzyme is the addition or removal of a functional group to or from the protein enzyme. This modification can alter the enzyme function - either activating or inhibiting it. Secondary enzymes are often responsible for the modification and provide a second layer of regulatory control over enzyme function.

When O2 binds to heme in hemoglobin, the ____ ion is drawn into the plane of the ____ causing a conformational change that is transmitted to adjacent subunits enhancing the ____ for additional O2 binding. A) Mg2+; globin; planarity B) Fe3+; heme; folding C) Mg2+; globin; attraction D) Fe3+; porphyrin; affinity E) Fe2+; porphyrin; affinity

E) Fe2+; porphyrin; affinity

Non-covalent interaction with small molecules (metabolites)

Enzymes may have the ability to bind certain small molecules at some specialized location in the enzyme, separate from the active site. Binding of these molecules can activate or inhibit the function of the enzyme. These molecules are called effector molecules. This form of regulation of the enzyme is called allosteric regulation ("allo" means "other"). The effector molecule binds at some site other than the active site of the enzyme and is structurally different from the substrate molecule. The binding interactions are non-covalent and thus reversible. The effector molecule may be a product or reactant at some downstream or upstream location in a chemical pathway that involves the enzyme being regulated (Feedback inhibition or activation)

AMP + glycogen phosphorylase

Glycogen phosphorylase conforms to the MWC model of allosteric transitions, with the active form of the enzyme designated the R state and the inactive form denoted as the T state (Figure 15.16). Thus, AMP promotes the conversion to the active R state, whereas ATP, glucose-6-P, and caffeine favor conversion to the inactive T state.

What is the defining characteristic of a reversible inhibitor?

It inhibits enzyme activity when bound to the active site, but activity is regained when the inhibitor is released.

Symmetry model p3

Most of the enzyme will be in a homogenous T state in the absence of bound effector molecules, meaning the equilibrium is shifted far to the left. The R state is assumed to have a much higher affinity for the substrate so substrate only binds to the R state. Once it binds this shifts the equilibrium such that more enzyme shift to the relaxed state. The binding of substrate is therefore a cooperative event.

What kind of effector is 2,3-Bisphosphoglycerate?

Negative Heterotropic effector

What type of inhibitor binds to an enzyme but not at the active site?

Noncompetitive inhibitor

sigmoidal curve

Plots of [S] vs reaction velocities (v) S curve

In Hemoglobin a tetrameric protein , once an oxygen molecule binds to the first subunit the structure changes such that the other subunits more readily take up more oxygen. What kind of allosteric model describes this mechanism?

Positive homotropic effector

6 main methods by which enzymes activity is controlled

Product Accumulation (Feedback) Substrate Depletion Genetic Regulation Allosteric Regulation Covalent Modification Zymogens and Isozymes

hemoglobin molecules play an important role in transporting CO2 in the opposite direction

- N-terminal amino groups of the Taut form of hemoglobin are available for reaction with CO2 and about 15% of the CO2 formed in tissues is carried to the lung covalently bound to the N-terminal nitrogens as carbamate - In the lung the high oxygen partial pressure favors formation of the Relaxed form resulting in reversal of the carbamate with release and exhalation of the CO2

What is the result of covalent modification of the Glycogen Phosphorylase enzyme?

- Phosphorylation of the enzyme causes it to change into the R form - Phosphorylation of the enzyme makes it less susceptible to feedback inhibition - Phosphorylation of the enzyme is reversible

Zymogen Examples

- The digestive enzymes are synthesized as zymogens in the pancreatic acinar (secreting) cells and stored as zymogen granules - Enteropeptidase catalyzes the activation of trypsinogen as it enters the duodenum - Trypsin catalyzes the activation of other zymogens - Trypsin action involves the peptide bond at the C- terminal side of lysine or arginine side chain.

Myoglobin Properties

- a single polypeptide chain of 153 amino acids - a single heme group in a hydrophobic pocket - 8 regions of α-helix; no regions of b-sheet - most polar side chains are on the surface - non-polar side chains are folded to the interior - two His side chains are in the interior, involved with interaction with the heme group - Fe(II) of heme has 6 coordinates sites; 4 interact with N atoms of heme, 1 with N of a His side chain, and 1 with either an O2 molecule or an N of the second His side chain

Hemoglobin Properties

- a tetramer of two a-chains (141 amino acids each) and two b-chains (146 amino acids each); a2b2 - each chain has 1 heme group; hemoglobin can bind up to 4 molecules of O2 - binding is cooperative (Allosteric); when one O2 is bound, it becomes easier for the next O2 to bind - the function of hemoglobin is to transport oxygen - the structure of oxygenated Hb (Relaxed) is different from that of deoxygenated Hb (Taut) - H+, CO2, Cl-, and 2,3-bisphosphoglycerate (BPG) affect the ability of Hb to bind and transport oxygen

Symmetry model p4

A key point of this model is that the enzyme is an oligomer and as such has a number (2+) of monomer units that can each bind and convert substrate. Conversion from taut to relaxed of 1 enzyme means that actually several sites for catalysis open up which explains the cooperative nature of the observed kinetics.

The subunits in the _______ conformation will bind to an activator and will _________ binding to the substrate. A) R; increase B) T; increase C) R; decrease D) T; decrease

A) R; increase

Which of the following statements regarding enzyme regulation is true? A) the conversion of trypsinogen to trypsin is an example of zymogen activation B) allosteric effectors are always more powerful than covalent modification C) addition of an inhibitor to a V system results in kinetics similar to addition of a competitive inhibitor to a typical hyperbolic system D) the T state of an enzyme generally has more activity than the R state E) none of the above

A) the conversion of trypsinogen is an example of zymogen activation

Effect of substrate depletion

Reaction velocities are proportional to the rate constant and the substrate concentration(s). The reaction rate will therefore decrease as substrate is depleted.

(R State) of the symmetry model

Relaxed

The structure of the binding site of oxygen in Relaxed Hemoglobin is

Relaxed

Forms of regulation by covalent modification

Reversible Phosphorylation Adenylation Uridylylation ADP-ribosylation Methylation Oxidation-Reduction Acetylation

(T state) of the symmetry model

Taut

Genetic controls over the number of enzyme molecules

The gene that codes for the enzyme can be regulated with regard to the number of copies of corresponding mRNA that are produced (i.e. transcribed), and therefore the number of enzyme molecules that are produced is controlled.

Other types of control over enzyme action: Zymogens

The zymogen form is typically a longer polypeptide that must be hydrolyzed at a specific location (or locations) to produce the active form of the enzyme. This proteolytic cleavage can be at either termini, or at some internal location in the polypeptide. The site(s) of cleavage are known as "activation sites". Cleavage at the activation site(s) may release a polypeptide known as the "pro-sequence", and results in activation of the enzyme. Cleavage is thus a regulatory mechanism of enzyme activity, and the protease(s) responsible for activation are therefore regulatory proteases, and are typically quite specific with respect to the sequence they cleave.

Glycogen phosphorylase regulation

When concentrations of ATP or glucose-6-P accumulate to high levels, glycogen phosphorylase is inhibited; when [ATP] and [glucose-6-P] are low, the activity of glycogen phosphorylase is regulated by availability of its substrate, Pi

Symmetry model p2

When not bound by an effector molecule the enzyme monomer units are described as R0 and T0 and once bound the units are R1 and T1. The R0 and T0 states exist in equilibrium

Myoglobin

a monomeric heme proteinfound mainly in muscle tissue where it serves as an intracellular storage site for oxygen. During periods of oxygen deprivation oxymyoglobin releases its bound oxygen which is then used for metabolic purposes.

Regulatory enzymes

an enzyme in a biochemical pathway which, through its responses to the presence of certain other biomolecules, regulates the pathway activity

covalent phosphorylation

conversion of phosphorylase b (less active) to phosphorylase a (more active) R state

2,3-bisphosphoglycerate (2,3-BPG)

derived from the glycolytic intermediate 1,3-bisphosphoglycerate, is a potent allosteric effector on the oxygen binding properties of hemoglobin. - like increased hydrogen ion concentration, increased 2,3-BPG concentration favors conversion of R form Hb to T form Hb and decreases the amount of oxygen bound by Hb at any oxygen concentration.

Hemoglobin

emeprotein found in erythrocytes where it is responsible for binding oxygen in the lung and transporting the bound oxygen throughout the body where it is used in aerobic metabolic pathways. Each subunit of a hemoglobin tetramer has a heme prosthetic group identical to that described for myoglobin.

Regulation of Enzyme activity

enzymes can be regulated by other molecules that either increase or reduce their activity. Molecules that increase the activity of an enzyme are called activatory, while molecules that decrease the activity of an enzyme are called inhibitors.

Isozymes

enzymes that can exist in multimeric forms, such as tetramers but can also have isomers of those forms that perform different catalytic activities. Isoforms of an enzyme differ slightly in amino acid sequence so they have different catalytic properties. The ability to "change out" the different monomer domains in the Isozyme allow it to be specialized or regulated to only work on the tissue it will serve.

Carbon dioxide affects O2 binding to Hb by: A) Hb competing with carbonic anhydride for CO2. B) directly binding to heme-Fe in the oxygen binding site. C) forming iron carbonate with the heme-iron. D) forming H+ + HCO3− where the H+ is an antagonist to oxygen binding to Hb. E) forming HCO3− that combines with H+ to increase CO2 binding.

forming H+ + HCO3− where the H+ is an antagonist to oxygen binding to Hb.

glycogen phosphorylase reaction

involves phosphorolysis of the bond between C-1 of the departing glucose unit and the glycosidic oxygen, to yield glucose-1-phosphate and a glycogen molecule that is shortened by one residue

Zymogen

precursor enzymes that require modification before becoming active. They are also known as a pro-enzyme..

symmetry model of allosteric behavior

the allosteric enzyme has an oligomeric structure in which all of the molecules adopt the same conformation (I.e. they are homogenous with regards to their monomer structures).

An allosteric effector molecule binds to ______ and cause a change in ___________.

the allosteric site; conformation of the enzyme

cooperative effect

the binding of a substrate molecule to one enzyme actually promotes the binding of substrate to another enzyme (Sigmoidal curve)

Glycogen phosphorylase

the enzyme that cleaves glucose from the ends of the glycogen polymer. This cleavage is a phosphorolysis process and produces a phosphorylated glucose molecule (a-D-Glucose -1-phosphate). - two substrates: phosphate ion, and glycogen. The binding of phosphate ion is cooperative. Phosphate (Pi) is therefore considered a positive homotropic effector.

feedback inhibition

the final product of a metabolic pathway is an allosteric inhibitor of the first enzyme in the pathway. This means that as product accumulates it slows or stops its own production. As it is consumed and need for the product arises again the production can resume.

Which of the following is true regarding effectors of oxygen-hemoglobin binding?

the presence of CO2 enhances the release of oxygen from hemoglobin

amplification cascade

what occurs when a single protease activates multiple copies of a zymogen and is itself a zymogen.