Where Effectors Bind Effector where does it bind? At the Active Site substrate product competitive inhibitor irreversible inhibitor At another site "designed site" allosteric inhibitor allosteric activator irreversible inhibitor "incidental site" noncompetitive inhibitor irreversible inhibitor Uncompetitive inhibitor

Inhibitors - Simple Enzymes ä ä Competitive Inhibitor - Has a shape similar to that of the substrate and binds to the active site of the enzyme and prevents the substrate from binding. ä ä Noncompetitive Inhibitor - Binds at a another site on the enzyme other than the active site ä ä not dependent on substrate binding ä ä not the same as allosteric! ä ä Uncompetitive Inhibitor - Binds at a another site on the enzyme other than the active site ä ä dependent on substrate binding ä ä not the same as allosteric ä ä Irreversible Inhibition - Occurs when an inhibitor forms a covalent bond to the enzyme and inhibits its activity.

Competitive Inhibition I Inhibitor and substrate have portion of shape in common Compete for active site Large amounts of inhibitor swamp out substrate binding and product formation is suppressed Likewise large amounts of substrate swamp out inhibitor binding and product formation is minimized

Competitive Inhibition II As the amount of inhibitor increases, there is more competition at the active site most pronounced at low levels of S At higher [S], inhibition is “swamped out” V max does not change processing rate not effected “K m ” increases!!! the enzyme does not bind the substrate as well in the presence of inhibitor [I] = 0 [I] = x[I] = 2x

Competitive Inhibition III [I] = x [I] =2x A Lineweaver-Burk plot allows the important parameters, K m and V max, to be determined directly from an equation rather than from an extrapolation 1/V max = y intercept K m /V max = slope of line Get V max first, don’t forget inverse relationship!!! Highest line most inhibited!!!! [I] = 0

Noncompetitive Inhibition I Inhibitor and substrate DO NOT have common shape Binding is NOT at active site but causes a change in the substrate binding site Large amounts of inhibitor prevent access of substrate to binding site Large amounts of substrate CANNOT overcome inhibitory effect

Noncompetitive Inhibition II As the amount of inhibitor increases, the efficiency of processing the product decreases same relative effect at all levels of S Higher [S] cannot “swamp out” effect; the “problem” is not at active site K m does not change binding/dissociation not effected “V max ” decreases the enzyme is less efficient in processing product [I] = 0 [I] = 2x [I] = x

Noncompetitive Inhibition III A Lineweaver-Burk plot allows the important parameters, K m and V max, to be determined directly from an equation rather than from an extrapolation 1/V max = y intercept K m /V max = slope of line Get V max first, don’t forget inverse relationship!!! Highest line most inhibited!!! [I] = 0 [I] = 2x [I] = x

Uncompetitive Inhibition I Inhibitor and substrate DO NOT have common shape Binding is NOT at active site Binding of substrate causes a change in the shape of the enzyme that allows inhibitor to bind Inhibitor binding prevents processing of substrate to product Larger amounts of substrate cannot overcome inhibitory effect

Uncompetitive Inhibition I As the amount of inhibitor increases: the binding is effected the efficiency of processing product decreases K m increases binding/dissociation effected “V max ” decreases the enzyme is less efficient in processing product ä BC Online: 6B - Models of Enzyme Inhibition BC Online: 6B - Models of Enzyme Inhibition

Controls of Enzymatic Activity ä ä Inductive effects ä ä changing the amount of active enzyme present ä ä genetic control; irreversible covalent modification ä ä Regulatory effects ä ä changing the activity of enzymes already present ä ä LeChatelier control; allosteric control; reversible covalent modification; hormonal control

Inductive Effects ä ä Genetic control ä ä Turn genes on  make mRNA  make enzymes ä ä Irreversible covalent modification ä ä Proteolytic activation ä ä zymogen ----> active enzyme ä ä very common for digestive enzymes

Regulatory effects ä ä LeChatelier ä ä every system responds to the levels of substrates and products ä ä Allosteric ä ä feedback: product of pathway providing information about the status of metabolism ä ä forward activation: the product of an earlier reaction telling a later enzyme to get ready ä ä Reversible covalent modification ä ä reversible phosphorylation usually on serine ä ä extra ramping up of enzymatic activity, sometimes related to hormonal response; control points in metabolic pathways ä ä Hormonal control ä ä control from a distance (on organism level) ä ä binding to outside of cell OR entry into cell causes effect

Feedback Inhibition ä ä “Information” from a later step in pathway ä ä Usually product of downstream reaction ä ä Amount of product has to build up before effect is “felt” ä ä Utilization of downstream product decreases effect

Forward Activation ä ä “Information” from an earlier step in pathway ä ä Usually substrate of upstream reaction ä ä Signal that lots of stuff is on the way ä ä Amount of substrate has to build up before effect is “felt” ä ä Utilization of upstream substrate decreases effect

Factors Influencing the Rates of Enzyme-Catalyzed Reactions ä Concentration of the substrate Rate Substrate Concentration Rate versus Concentration of Substrate Most Enzymes Rate Substrate Concentration Rate versus Concentration of Substrate Allosteric Enzyme

Allosteric Regulation of Enzyme Activity ä ä Allosteric Enzymes - Enzymes composed of two or more protein chains which contain separate regulatory sites and active sites. ä ä Often the regulatory site is on one chain and the active site on another. ä ä Binding of a molecule at the regulatory site changes the shape of the enzyme and affects its activity. ä ä Activator - a positive regulator ä ä Allosteric inhibitor - a negative regulator ä ä not the same as noncompetitive inhibition

Concerted Model of Allosteric Effect T T R R L The substrate,, can only bind to the Relaxed (R) form of the enzyme The Taut (T) and Relaxed (R) forms are in equilibrium with each other with an equilibrium constant value of L L = [R]/[T] The value of L is much less than 1 As the concentration of increases, equilibrium is shifted to the right Since L remains constant, more of the T form is converted to the R form and there is more enzyme in the R form and the reaction speeds up R R

Sequential Model of Allosteric Effect T T R R L The substrate,, can bind to both the Relaxed (R)and Taut (T) forms of the enzyme The R form is bound preferentially Only the site that has bound the substrate changes conformation by induced fit The binding of the initial substrate passes on the conformational change to other subunits These units will now bind the substrate with greater ease As the concentration of increases, the number of relaxed active sites increases and the reaction speeds up sigmoidally form and the reaction speeds up R R R T

“Sense” of Cooperativity ä ä every protein is in its lowest energy configuration given the prevailing conditions ä ä the "resting" form of an enzyme is in the taut form ä ä either in equilibrium with relaxed form ä ä or can be changed to the relaxed form when substrate is added (or increased) ä ä this is the “turtle in its shell mode” -it is safe ä ä the relaxed form becomes more favored upon the addition of substrate (or activator) ä ä the prevailing conditions have changed! ä ä “turtle in its moving and eating mode”- also more vulnerable!