P1 Biochemistry Enzyme Regulation

Allosteric Modulation Occurs at a "Distant" Site Allosterism involves the binding of a ligand (effector) at an allosteric (allosteric . other shape) site. Positive allosteric effectors?? Negative allosteric effectors: Ligands??

: Ligands stabilize the relaxed (more active) form; activation is seen as sigmoidal curve displaced to the left : stabilize the tense (less active) form; inactivation is seen as sigmoidal curve displaced to the right

Allosterism - Effect on Kinetics Allosterically-regulated enzymes do not follow the Michaelis-Menten equation; the kinetics of interaction are different. A positive allosteric substance converts the enzyme from the _______ to the _____ A negative allosteric substance converts the enzyme from the ____ to the _____ Allosteric modulation is reversible, which allows the modulator molecule to provide a rapid action in the enzyme pathway that can be terminated quickly as needed.

: T (tense form) to the R form (relaxed) : R form (relaxed) to the T form (tense)

Changing the Activity of Enzymes: Proteolytic Activation Proteolytic activation means that ?? Most proteins fold into their active conformation upon synthesis. However, some enzymes are first synthesized in an inactive form, termed a _____ or a _____ and stored in a "_____" until needed. Activation occurs upon ...? The digestive enzyme _____, is a good example.

: an inactive precursor protein is changed chemically to produce active enzyme; irreversible covalent modification. : zygomen : proenzyme : zymogen granule : hydrolysis of one or more peptide bonds, allowing the protein to fold into a new and active conformation. : chymotrypsin

Control Proteins: Calcium-Dependent Enzymes A rapid rise in intracellular calcium is often a signaling event in many types of cells. A small 17,000 MW protein named _____ binds the available Ca++ and the bound calcium causes calmodulin itself to _____ This binding of calcium causes calmodulin to interact with various target enzymes and induce a conformational change in those targets. The change does what?? So rising levels of calcium in the cell triggers the control protein, calmodulin, to interact with various enzyme targets and dramatically affect cellular functions.

: calmodulin : become active : converts the target enzyme from an inactive form to an active form.

Example of Allosterism - ATCase ATCase catalyzes the ...? An end product of the pathway, ...? This feedback inhibition signals that there are ...? ATP (a purine nucleotide) is a _______ allosteric effector of ATCase; an _____ of ______ signals the need for more _____ to balance the purines ATCase is a _____ protein containing both catalytic (C) and regulatory (R) subunits (C6R6).

: committed (first) step of pyrimidine synthesis in E. coli, e.g. the nitrogenous bases Thymine, Cytosine and Uracil. : cytidine triphosphate (CTP; made 6 steps later), binds to a special subunit of the ATCase enzyme and inhibits this step - "feedback inhibition." : sufficient molecules of CTP being made to satisfy the needs of the cell for pyrimidine nucleotide. : positive : excess : ATP : pyrimidines : purines : multi-subunit

Allosteric Modulation Occurs at a "Distant" Site Allosterism is a response to an effector molecule (endogenous/physiological metabolite) by an enzyme that results in an increase or decrease in its activity. The enzyme must be ______ to show allosterism As seen with hemoglobin, cooperativity is a result of communication between subunits (monomers) of the enzyme quaternary structure. The binding of a ligand induces a change in conformation in adjacent subunits that increases the binding of the additional ligand on the adjacent subunits Cooperativity creates S-shaped (sigmoid) substrate versus velocity plot

: cooperative

Reversible Covalent Modification - Acetylation of Lysine side chains in Histones Another important example of reversible covalent modification is the acetylation and deacetylation of specific Lys side chains in histone proteins. Histones are basic proteins that associate and disassociate with DNA and are integral to chromatin structure. These reversible modifications of DNA play an important role in regulating gene transcription. Acetylation of histones does what?? Deacetylation of histones does what??

: decreases their interaction with DNA, which opens up the chromatin (euchromatin) and allows transcription to proceed; acetylation "allows" gene expression to proceed. : causes the protein to bind more tightly to DNA and increase the condensation of the chromatin (heterochromatin), thereby preventing transcription; deacetylation "deactivates" or silences DNA.

______ different from regulation, which occurs under the influence of ______ A non-physiologic agent (e.g., ______) may cause enzyme inhibition

: inhibition : normal cellular metabolites. : drugs and toxins

What are the advantages of this cascade approach to coagulation? One advantage of the proteolytic coagulation cascade and the use of pre-existing zymogens is that ...? Clotting is a response to injury so the factors remain inactive until needed. If the factor was active at the wrong time. it could trigger clot formation, which would impair blood flow in normal healthy tissue. Normal, undamaged tissue secretes anti-clotting factors that modulate activity and hold the system in check (see the "red" pathways on the coagulation cascade diagram). A second advantage is that ...? In each proteolytic step of the cascade shown, a single protease may activate hundreds or more molecules resulting in an exponential amplification of activated factors over many orders of magnitude. This amplification is explosive and contributes to the rapid formation of the clot. However, fibrin production is ...?

: it may be rapidly deployed. The factors are already present in the blood and ready at a moments notice. : the cascade is also an amplification system. From only a few molecules indicating damage is present, a sequence is initiated that leads to a macroscopic fibrin clot. : irreversible - no way to get back to fibrinogen - so the liver continuously makes the protein zymogens of the clotting cascade.

Techniques to Regulate Enzymes The amount or distribution of enzyme molecules can change (ET) - however, the time course for the effect may be too slow 1)We can increase the amount of enzyme by _____. Also termed ______ synthesis or increased ______. Requires time to make _______. 2) There can be increased _______ or ______ of the enzyme (enhanced breakdown). [___] is lower. 3) Compartmentation of the enzyme. An enzyme that is found only in the ________, for example, can only work on ______. This can also effectively ...?

: making more : de novo : gene expression : new protein : destruction : turnover : E : mitochondrion : substrate present in that location : raise or lower [E] in the local area.

Coagulation Cascade The coag cascade is another example of ...? If a blood vessel ruptures or leaks inappropriately due to some issue, the leak must be sealed rapidly. In response, the blood protein fibrin aggregates in the opening and traps platelets, other blood cells and large proteins, forming a plug or clot. There is no time to ...? Blood clotting must be tightly controlled - the cascade operates on a knife edge of balanced enzyme activity. Too little activity and bleeding may go unrecognized. Too much activity and inappropriate clotting may occur in vessels (thrombus) or a clot may break away (embolus) and lodge somewhere like the brain, causing a stroke.

: proteins synthesized in inactive states that are activated upon proteolysis. : synthesize fibrin, it must be pre-existing in the bloodstream as a zymogen precursor, fibrinogen.

Graphical look at change in enzyme activity with allosterism present Allosterism causes the hyperbolic curve to be sigmoidal (like Hb) Excessive pyrimidines (CTP) cause the enzyme to be inhibited (_____ shift), while excessive purines (ATP) cause the enzyme to be activated (note the _____ shift); this restores the balance between the nitrogenous bases Apparent Km changes - ?? Enzyme conformation (relative percent of __ vs.____) has been changed Vmax reached?

: right : left : affinity of the enzyme for the substrate is different : T vs R state : can be reached given enough substrate

Changing the Activity of Enzymes: Reversible Covalent Modification- Activated Protein kinase A is an example Many enzymes can be reversibly modified by _____, the most common of which is ______. Activated Protein kinases catalyze ...? This does what? Phosphorylation may result in either activation or inactivation of the targeted enzyme. Importantly, phosphorylation is _____!. An enzyme termed a phosphatase ..? An enzyme can cycle between the active and inactive form many times, and rapidly, in response to changing physiological conditions.

: small functional groups : phosphorylation : the transfer of a phosphate group from the terminal phosphate of adenosine triphosphate (ATP) to typically a serine or threonine side chain hydroxyl function if contained in certain primary amino acid sequences of the target enzyme. : introduces abulky group with 2 negative charges onto the enzyme, t hus leading to significant conformational effects. The phosphate can form ionic interactions or 3 hydrogen bonds. : reversible : can remove the phosphate from the side chain and return the enzyme to the original state.

Changing the Activity of Enzymes: Control Proteins The activity of many enzymes is controlled by interaction with other proteins in a cell - activity may be enhanced or inhibited - the essence of "regulation" that is our topic Such proteins are termed control proteins Examples include ...?

: transcription factors, plasma membrane channels and receptors and enzymes regulating calcium transcription factors, plasma membrane channels and receptors and enzymes regulating calcium

After the bleeding . . . The fibrin clot formed at the leak is simply a bandage over a wound. Tissue repair processes will fix the blood vessel and surrounding tissue. Then the clot must be removed so that normal blood flow and function of the affected area may resume. Clot removal also involves ______. The damaged tissue releases tissue plasminogen activator (TPA) during the repair. TPA works how?? TPA has been developed as a successful biotech drug that is now often used to dissolve clots, such as those that form after myocardial infarction or thromboembolic stroke. Quick use of TPA has resulted in much improved clinical outcome.

: zymogen activation : activates plasminogen (a blood protein zymogen added to the clot as it forms) to plasmin. Plasmin cleaves fibrin into soluble peptide fragments, thus dissolving the clot.

Some key points regarding the Coagulation Cascade Fibrinogen/Fibrin, Prothrombin/Thrombin and Factor X Two separate coagulation pathways feed into a common system. Each consists of a series of proteins that are almost al_____ that are proteolytically activated by tissue factors released either by internal vessel damage (intrinsic) or by external tissue trauma (extrinsic). The two pathways join in the common pathway with the activation of Factor X to Xa So Factor Xa is produced by either (or both) of the intrinsic pathway or the extrinsic pathway. Activated Factor X (Xa) proteolytically cleaves (activates) Prothrombin (II), which is converted to Thrombin (IIa). Thrombin IIa stimulates the enzyme Thrombin protease to convert Fibrinogen (I) to Fibrin (Ia), and fibrin traps proteins, cells and other materials in a mesh network to form the clot and stop the bleeding.

: zymogens

3. Which of following proteins is a regulatory protein that affects enzyme activity through direct protein to protein interactions. A. calmodulin B. aspartate transcarbamoylase C. protein kinase A D. plasmin

C

2. Regarding allosteric modulation of enzyme activity, A. allosteric modulators bind to the active site of the enzyme. B. negative allosteric modulators decrease the Km for the substrate. C. positive allosteric modulators cause a conformational change from the R to the T state. D. show a sigmoidal v vs. [S] plot.

D

4. Protein kinase A A. causes irreversible activation of its protein substrates. B. uses cyclic AMP as the phosphate donor. C. transfers a hydroxyl group to an ATP molecule in its protein substrate. D. may cause either enhanced or decreased activity of its target enzyme protein.

D

5. A blood clot consists of a giant protein precipitate of what protein? A. thrombin B. factor X C. plasmin D. fibrin

D

6. Allosteric inhibition is different from pure Michaelis-Menten noncompetitive inhibition because allosteric inhibition can be shown to produce: A. Unchanged substrate affinity B. Lower apparent Km C. Linear Rate vs. [S] plot D. A distinct "right shift" of the Rate vs. [S] plot

D

So is Allosterism "Competitive" or "Noncompetitive" Inhibition??

Short answer: NO, it is neither !!! Allosterism does not follow M-M kinetics Rate vs. [S] plots will be sigmoidal, not hyperbolic L-B plot will not be linear Although Km is changed and Vmax constant like competitive inhibition, the site of allosteric binding is not at the catalytic or true substrate binding site, thus allosterism is more like noncompetitive inhibition Remember however, M-M noncompetitive inhibition causes a change in Vmax with no alteration of Km So keep it clear; do not confuse allosteric modulation with the main types of inhibition that follow M-M kinetics Allosterism changes the conformation of the enzyme subunits to affect substrate binding and product formation

Rate Limiting Step

The enzyme that catalyzes the slowest step in a pathway is termed the rate-limiting enzyme. The rate-limiting enzyme may or may not be the same as the major regulated enzyme. From a pharmaceutical point of view, the rate-limiting enzyme is the most desirable target for inhibition. A 50% inhibition of the rate-limiting enzyme is going to shut down the pathway 50%, but a 50% inhibition of a rapid step may have no effect at all on the flux through the pathway.

Committed Step

The first step in the pathway that is unique to the pathway (and usually irreversible) is called the committed step. In general, the enzyme catalyzing this reaction is the most important point for regulation. An example is the conversion of fructose-6-phosphate to fructose-1,6-bisphosphate by phosphofructokinase in the pathway of glycolysis. You will see other examples as we cover metabolic pathways.

Feedback Inhibition

When the product of a pathway inhibits a prior step in the pathway, this type of control is termed feedback inhibition. For example, pyrimidine nucleotides (e.g. CTP), which make up part of the structure of DNA and RNA, turn off their own synthesis when the levels are sufficiently high to satisfy their role in the cell.

Techniques to Regulate Enzymes, con't Change the "_____" of existing enzyme There are a number of mechanisms used in biochemistry to up- or down-regulate the activity of enzymes - these mechanisms are more rapid: 1) Allosteric Modulation 2) Control Proteins 3) Reversible Covalent Modification 4) Proteolytic Activation

activity

The top view of ATCase The quaternary structure of ATCase as viewed from the top. A single catalytic trimer [(c) chains, shown in yellow] is visible; the second trimer is hidden below the one visible. Notice that each r chain interacts with a c chain through the _____ domain

zinc