Review Reaction mechanisms 2. Amino acid mutations and their effects Problem Set #3 Quiz #3

Review Reaction mechanisms Draw the catalytic amino acid side chains with an eye for geometry Remember the substrate and products Use reaction arrows to move to another panel when you need to For this class, focus on Attack! Electrons and Lone Pairs are the players here, not positive charges or protons Let’s go through the serine protease mechanism again.

Review Amino acid mutations A mutation is not an inhibitor!!!!! What are the effect(s) of the mutation? Which kinetic parameters change? What does that tell you? Can you relate it to what we already know about kinetics? You must think about the relationship between the actual mutation, the physical difference between the wild type and the mutant as well as the effect of this difference

Quiz #3 Problem #1: Most of you didn’t read the problem 1) Proteases in the extracellular matrix frequently use divalent metal cations in addition to amino acids to catalyze the hydrolysis of peptide backbone. Analysis of a new matrix metalloprotease found the following: a. A zinc ion, an arginine and a glutamate are the key catalytic residues for the protein b. The preferred substrate is a dipeptide, Gly-Tyr. c. The zinc ion is involved in an ion-dipole interaction with the oxygen atom of water. d. The water molecule bond by the zinc ion is in close proximity to the carboxylate of the catalytic glutamate. e. The pH of the active site is 7.2.

Quiz #3 Problem #1: How do you solve it? READ WHAT YOU ARE GIVEN!!!!! Remember the rules for reaction mechanisms Protons don’t attack anything You must regenerate the amino acids What are the charges on amino acids at physiological pH? THE ELECTRONS DO THE ATTACKING Draw lone pairs and charges

Quiz #3 Problem #2: Most of you didn’t read the problem 2) The following questions deal with a mutagenic study of a protein. Explain the effect of the mutations in terms of the reaction equation and the chemical structure of the active site or protein. a. You mutate a serine in the protein to an alanine. When you perform a kinetic analysis of the mutant protein and compare the Lineweaver-Burk plot to that of the native protein, the x-intercept changes, but the y-intercept does not. What has the mutation done? b. You mutate a glutamate to a glutamine. When you perform a kinetic analysis of the mutant protein and compare the Lineweaver-Burk plot to that of the native protein, the y-intercept changes, but the x-intercept does not. What has the mutation done?

Quiz #3 Problem #2: How do we solve it? Do you even know what is happening? What is the difference between an inhibitor and a mutation? What does a Lineweaver-Burk plot tell you? What do the kinetic parameters of an enzyme mean? If they change, what does that tell you about the REAL, STRUCTURAL and CHEMICAL effects of the mutation?

Problem Set #3 Problem #1: This one was tough. For me. This is the first enzyme we looked at. The full mechanism is in your book. I have drawn it on the board several times (most recently for Lecture 16) All the mechanism rules apply What is the substrate and what are the products? Nucleophilic attack (Lone pairs and/or negative charges do the work) Serine does the work after Histidine activates it. Remember the charge of amino acids at physiological pH

Problem Set #3 Problem #1: How do we solve it? As part of your senior research project, you have identified a novel bacterium that is capable of degrading the coat protein of the H1N1 virus. The protease responsible for the antiviral effect hydrolyzes the peptide bond between phenylalanine and alanine in the substrate. Analysis of the protein shows that it is a serine protease. Draw the reaction mechanism for this reaction. You must show all atoms of the substrate and the side chain atoms of the catalytic amino acids in the enzyme in your answer. You must clearly show the arrows indicating the formation/dissolution of bonds in your answer.

Problem Set #3 Problem #2: Not many problems with this one, except for Sucrose 1) Draw the Haworth projections of: a. Maltose b. Lactose c. Cellotriose d. Sucrose

Problem Set #3 Problem #2: How do we solve it? Write the arrangement out before you start drawing structures. Draw the monomers Draw the linkage. Sucrose is the problem child: Glucose (-1,2) Fructose

Problem Set #3 Problem #3: Forget about it I’m dropping the 10 points from the total for the problem set. You keep all of your points

Problem Set #3 Problem #5: Here we go again… Mutation of the catalytic glutamate in chicken egg white lysozyme to an aspartic acid results in a kcat decrease of 75% relative to the native enzyme. a. What is the value of Vmax for the mutant enzyme relative to the native enzyme? b. Do you expect the mutation to have any effect on the Km for the enzyme? Why or why not? Explain the effect of the mutation by drawing the active site of the native and mutant forms of lysozyme and comparing them. If you had mutated the catalytic aspartic acid known to be involved in the reaction mechanism of lysozyme to an asparagine, what effect would you expect on Vm and Km for the enzyme? Use everything you can to prove your point (drawings, equations and logic).

Problem Set #3 Problem #5: How do we solve it? There are no inhibitors! Nearly everyone said that there were. Why? Take what is given to you and think about it. This is the hard part. What data are you given? What do those data mean in reality?

What have we learned? Read the problems. There is no excuse for not reading what you are given and using it. None. Read your notes and your textbook. Many of you ignored what had been discussed in Lecture 16 What are the reactants, products and active site residues? Draw everything out with plenty of room. GO STEP BY STEP! Stop running for the finish line straight away, you’ll leave out details that are important Check your work!