lecture 11.105 Inhibitor kinetics, lipids

Biological Sciences 105 Lecture 11, November 6, 2017 Copyright Steven M. Theg, 2018. All federal and state copyrights reserved for all original material presented in this course through any medium, including lecture or print. 1 When we ended last time we had just finished talking about inhibitors. Competitive inhibition – last time         + + = I M MAX o K I K S S V v ] [ 1 ] [ ] [ See that         + = I M app M K I K K ] [ 1 , , so that app M MAX o K S S V v , ] [ ] [ + = The reciprocal form is MAX I MAX M V S K I V K v 1 ] [ 1 ] [ 1 1 0 + •                 + = So you can see that what has changed is the slope, not the y-intercept. The properties are these: As [I] increases, the reciprocal plot pivots about the y- axis intercept counterclockwise. The slope, K M /V max , increases, while the x-axis intercept decreases, as Km,app increases.

Biological Sciences 105 Lecture 11, November 6, 2017 Copyright Steven M. Theg, 2018. All federal and state copyrights reserved for all original material presented in this course through any medium, including lecture or print. 2 How do you find K I ? A number of ways. 1. You can look at the x-axis intercept for – 1/K M,app , and calculate K I from there. Use         + = I M app M K I K K ] [ 1 , , so K I = 𝐾 𝑀 [𝐼] 𝐾 𝑀,𝑎𝑝𝑝 −𝐾 𝑀 2. Given v 0 with I present, V MAX , KM, [I] and [S], solve for K I . I M M I M K I K K S S V K I K S S V v ] [ ] [ ] [ ] [ 1 ] [ ] [ max max 0 + + =         + + = ] [ ] [ ] [ max 0 0 0 S V K I K v K v S v I M M = + + M I M K v S v S V K I K v 0 0 max 0 ] [ ] [ ] [ − − = ( ) M M I K v S v V I K v K 0 0 max 0 ] [ ] [ − − = Now simply plug in the numbers. There other more complicated graphical ways.

Biological Sciences 105 Lecture 11, November 6, 2017 Copyright Steven M. Theg, 2018. All federal and state copyrights reserved for all original material presented in this course through any medium, including lecture or print. 4 Now K I has two equivalent expressions: ] [ ] ][ [ ] [ ] ][ [ EIS I ES and EI I E K I = = When this is not true, have mixed non-competitive inhibition. Probably more common, but the equations are more complicated. We’ll skip that here. The derivation is essentially the same as for competitive inhibition, but the K I is different. Start with v 0 = k cat [ES], then divide by E t = E + ES + EI + EIS, etc. When you’re done with the algebra, the equation will look so: ( )         + + = I M MAX K I S K S V v ] [ 1 ] [ ] [ 0 You get a better appreciation of where the effect of the inhibitor is when you group it as follows:         + •             + = M I MAX K S S K I V v ] [ ] [ ] [ 1 0

Biological Sciences 105 Lecture 11, November 6, 2017 Copyright Steven M. Theg, 2018. All federal and state copyrights reserved for all original material presented in this course through any medium, including lecture or print. 5 Now you see that when I increases, V MAX decreases, so that I MAX app MAX K I V V ] [ 1 , + = Look back at the reciprocal plot. As [I] increases, the slope again increases, but this time pivoting at the x-axis intercept. The reciprocal plot is (setting  = 1 + (I/K I ): max max max 0 ] [ 1 ] [ ] [ 1 V S V aK S S K V v M M   +  = +  = Now you see that what is different, or affected by  , is the slope and the y-intercept. What about x-intercept. Set y = 0, max max ] [ 1 0 V S V K M   +  = ] [ 1 max max S V K V M  = −  

Biological Sciences 105 Lecture 11, November 6, 2017 Copyright Steven M. Theg, 2018. All federal and state copyrights reserved for all original material presented in this course through any medium, including lecture or print. 7  ] [ ] [ max 0 S K S V v M + = Multiply both sides by  , then put this  in the denominator of the denominator. ] [ ] [ max 0 S K S V v M    + = ] [ ] [ ] [ ] [ max 0 S K S S K S V v M M + = + =     Move V max back, and write out  . ] [ ] [ 1 ] [ ] [ 1 max 0 S K I K S K I V v I M I +             +             + = Now we see that ] [ ] [ , max, 0 S K S V v app M app + = . Note, K M,app is different here than for competitive inhibition – it decreases.

Biological Sciences 105 Lecture 11, November 6, 2017 Copyright Steven M. Theg, 2018. All federal and state copyrights reserved for all original material presented in this course through any medium, including lecture or print. 8 The reciprocal plot looks like so: This is diagnostic of uncompetitive inhibition. How do you get K I now? Same thing. Look at K M,app or V max,app

Biological Sciences 105 Lecture 11, November 6, 2017 Copyright Steven M. Theg, 2018. All federal and state copyrights reserved for all original material presented in this course through any medium, including lecture or print. 10 Fatty Acids and Lipids Lipids: small biological molecules whose defining feature is their relative insolubility in water. Generally made of fatty acids and sterols. What do lipids do, and why are they important? 1. Principal form of stored energy in many organisms. The breakdown of lipids into fatty acids and sterols provides compounds used at the starting points of these pathways. 2. These are the principal components of membranes. The hydrophobic properties allow membranes to self-assemble. The hydrophobic properties also allow membranes to act as barriers between aqueous solutions. 3. Also important as second messengers; intracellular signals. One of the important pathways of signal transduction mentioned earlier uses lipid intermediates. Fatty Acids A fatty acid is a long chain hydrocarbon with a carboxylic acid at its end. The hydrocarbon can be from 4 to 36 carbons long. Because of the way fatty acids are synthesized, they are almost always found in multiples of two. Nomenclature is difficult. A C-12 fatty acid is lauric acid, C-16 is palmitic acid, etc. A more reasonable nomenclature is dodecanoic acid for C-12, hexadecanoic acid for C-16, etc. The simplest nomenclature is with numbers. First number is the number of C in the fatty acid. Followed by :#, with the second number being the number of unsaturated (double) bonds. For example, 20:2 is a 20 C fatty acid with two double bonds. There is a designation for the position of the double bonds.  # , i.e., C20:2(  9,12 ) is a 20:2 fatty acid unsaturated between C-9 and C-10, and again between C-12 and C-13. You should learn the last two nomenclatures. About the double bonds: The position of the double bonds is regular. A single double bond is almost always a t Δ 9 . Others are almost always Δ 12 or Δ 15 .

Biological Sciences 105 Lecture 11, November 6, 2017 Copyright Steven M. Theg, 2018. All federal and state copyrights reserved for all original material presented in this course through any medium, including lecture or print. 11 The double bonds are usually in cis, meaning they put a kink in the chain. Solubility and melting: Expect these to be soluble in water? No. Generally, the longer the hydrocarbon, the poorer its solubility. Why? What about a shorter fatty acid makes it more soluble than a long one? COO - . Long, saturated hydrocarbons pack together in a compact form. Stabilized by what kinds of interactions? van der Waals. What might the effect of an unsaturated bond be? Disrupts the packing, diminishes the vdW interactions. Should a saturated or unsaturated fatty acids have a lower melting point? Unsaturated. Triacylglycerols The simplest lipids formed from fatty acids are triacylglycerols. Also referred to as triglycerides, fats or neutral fats.

Biological Sciences 105 Lecture 11, November 6, 2017 Copyright Steven M. Theg, 2018. All federal and state copyrights reserved for all original material presented in this course through any medium, including lecture or print. 13 Waxes are the major storage material of some organisms. Waxes have a different (higher) melting point than triglycerides. This makes them (semi)- solid at relatively high temperatures. This property allows them to be used as an insulator against water. Beeswax, secretions on feathers of waterfowl. SKIP TABLE Lipids as components of membranes The lipids used in forming membranes are different than those used as energy reserves.

Biological Sciences 105 Lecture 11, November 6, 2017 Copyright Steven M. Theg, 2018. All federal and state copyrights reserved for all original material presented in this course through any medium, including lecture or print. 14 Glycerolphophoslipids have glycerol at the head group and fatty acids in tail, but also a phosphate-alcohol. The general structure of a phospholipid is: When X is: H, the lipid is phosphatidic acid When X is ethanolamine, phosphatidylehtanolamine When X is choline, phosphatidylcholine When X is serine, phosphatidylserine

Biological Sciences 105 Lecture 11, November 6, 2017 Copyright Steven M. Theg, 2018. All federal and state copyrights reserved for all original material presented in this course through any medium, including lecture or print. 16 Cholesterol is the major sterol lipid in animals. The OH group on the first ring makes it amphipathic. Aggregates formed from amphipathic lipids Class demonstration. What do you think would happen if you put a very small amount of lipid (which is an amphipathic molecule) into water? Experimentally, you get a tiny flake of dried lipid and touch it to the water surface. Where would the head group tend to go? Where would the tail tend to go? Answer: the lipids would form a monolayer across the surface of the water, with the head groups in contact with the water and the tails sticking up above the interface.

Biological Sciences 105 Lecture 11, November 6, 2017 Copyright Steven M. Theg, 2018. All federal and state copyrights reserved for all original material presented in this course through any medium, including lecture or print. 17 What happens when the amount of lipid added to the water exceeds the surface area of the water? Some is forced underneath. Now it depends on the type of lipid added. Single tail lipids form micelles, with the tails inward and polar heads out. Critical micelle concentration Lipids with two tails, the phospholipids and sphingolipids, form bilayers. You can shake the lipids in the solution and the bilayers will form into liposomes, capturing an aqueous phase in the interior. Now beginning to act like real membranes.