Catalysis & specificity: Proteins at work

Transcription

1 Catalysis & specificity: Proteins at work Introduction Having spent some time looking at the elements of structure of proteins and DNA, as well as their ability to form intermolecular interactions, it is now time to explore the structural basis of activity. In the case of enzymes, this activity is the ability to catalyze chemical reactions. We will also take the opportunity to look at another major class of proteins the membrane proteins. Objectives To understand the chemical basis for catalysis. To gain experience identifying the structural basis of activity. To work through examples of specificity and non-specificity. To explore membrane protein structure and compare/contrast with globular proteins. Brandén & Tooze 2 nd Edition: Chapters 11 & 12 Data files This lab uses files contained in the downloadable self-expanding archive Part 1 Exercise 1 Trypsin/inhibitor Trypsinogen is one of the pancreatic zymogens (precursor digestive enzymes). The pancreatic zymogens also include chymotrypsinogen and procarboxypeptidase. In their active forms, trypsin and chymotrypsin are members of the chymotrypsin superfamily, which also bon specific bonds within the peptide chain, they belong to the endopeptidases. Pancreatic trypsin inhibitor is a protein of 56 amino acids that bind very tightly to trypsin Kd = M. The surface area buried in the complex is some 1400 Å similar to the antibody-antigen complexes. The binding pocket in trypsin is actually formed by two separate regions of the backbone, residues from about and residues from The mechanism described below is illustrated in B & T pp In the first step of the cleavage reaction, the serine (195) attacks the carbonyl of the substrate peptide amino acid, forming a tetrahedral transition state. The OH of the Ser is hydrogen bonded with the imidazole ring of the histidine, which acts as a base, (proton acceptor). The transition state reforms the carbonyl double bond and breaks the carbonnitrogen bond of the amino acid, thus breaking the peptide bond. The amino portion accepts the hydrogen from the histidine and one part of the peptide chain breaks free. This is the acylation phase. The remaining carbonyl part, still attached to the serine, must be removed and the active site is regenerated. The second step removes the carbonyl part of the peptide amino acid from the serine through a reaction with water. Water reacts with the carbonyl group forming a second tetrahedral intermediate. When the carbonyl bond reforms, the bond to the serine oxygen is broken, freeing the second part of the peptide chain. The hydrogen bonded to the histidine is lost to the serine Oxygen and a hydrogen bond is reformed between

2 the serine and histidine. The third member of the triad, in both trypsin(ogen) and chymotrypsin(ogen) is an aspartic acid residue. The Asp residue, by forming a hydrogen bond with one of the nitrogens in the imidazole ring of the His residue, stabilizes, by polarization, the imidazolium ion formed when the ring accepts a proton from the serine residue. 1. Take a moment and work through the mechanism of base-catalyzed amide hydrolysis. 2. Take a look at the trypsin/bpti complex (2ptc.pdb) in cartoon format (script 2ptc_1.txt). Trypsin is orange (chain "e") and the inhibitor is yellow (chain "i") with disulfide bonds in green. What type of structures (fold) are they? The active site catalytic triad of trypsin is shown with the script 2ptc_2.txt. Notice how the fold brings these sequentially distant residues together. 3. What do the terms specific and non-specific mean in terms of Branden & Tooze s description? 4. Trypsin specifically cleaves after Lys and Arg side chains in polypeptides. The specific residue recognized in the trypsin inhibitor is Lys15. Find those residues on trypsin responsible for recognition of the Lys 15 side chain. The peptide bond is cleaved only very slowly (weeks!) in this inhibitor by the enzyme. Why should this be different than more general substrates? 5. Explain (using transition state theory) how enzymes function. Be sure to make diagrams to support your explanation. 6. Give two ways in which inhibitors may function. Be sure to make diagrams to support your explanation. 7. Structures determined of each protein alone demonstrate that the structure of both enzyme and inhibitor change very little upon complex formation (lock-and-key). How does this affect the thermodynamics of complex formation? 8. In trypsin, catalysis is facilitated by the stabilization of the transition state intermediate. This intermediate contains an oxyanion of the substrate scissile peptide bond (between 15 and 16 in the inhibitor). Try to identify this oxyanion hole in the 3D structure of trypsin. 9. Does the structure also provide the means for decomposing the acyl-enzyme intermediate? Or is it left to chance? Exercise 2 Subtilisin/inhibitor Subtilisins are a group of serine proteinases produced by different bacilli. These enzymes have found commercial use as additives to detergents. Subtilisn has no sequence homology to trypsin (you will recall the exceptional β α β connection), yet has a catalytic triad identical to trypsin. The bond cleaved by proteinase Subtilisn is after the specifically recognized Leu 45.

3 1. Take a look at the complex (1cse.pdb) in cartoon format (script 1cse_1.txt). Coloring is as in the trypsin example. What type of structure fold is this inhibitor? Do either of the enzyme or inhibitor have SS bonds? Display the active site catalytic triad of Subtilisn (script 1cse_2.txt). Notice how the fold brings these sequentially distant residues together. 2. Subtilisn specifically cleaves after hydrophobic residues (in this case Leu 45). Find those residues on Subtilisn responsible for this specificity. 3. Notice the interaction between the inhibitor residues and Subtilisn residues and What kind of interaction is formed? 4. Do solvent water molecules play a role in recognition? Catalysis? Part 2 Membrane protein structure/function Exercise 1 Bacteriorhodopsin The purple membrane of Halobacterium halobium contains a protein, bacteriorhodopsin, which binds retinal to harness light energy in order to pump protons across the membrane. The protein consists of 7 transmembrane helices aligned at an angle of about 20 with respect to the plane of the membrane. 1. Take a look at bacteriorhodopsin (1ap9.pdb). Identify the 7 helices and the retinal ligand. 2. Can you determine how the protein bacteriorodopsin interacts with its environment? Is it amphiphilic? If so how? Make use of the Java implementation of the hydrophobicity plot Plots.htm simply paste in the amino acid sequence. The amino acid sequence of the bacteriorhodopsin is supplied in file 1ap9_fasta.aa. 3. You may wish to use the helical wheel program from a previous lab to see if the helical segments are amphiphilic. Exercise 2 Porin Outer membranes of Gram-negative bacteria contain a protein called porin responsible for non-specific passage of small molecules from inside to outside. All molecules smaller than the inside of the barrel (9 Å long and 8Å in diameter) should be able to pass through. The biological unit of porin is a trimer of identical subunits.

4 1. Take a look at one subunit of the porin trimer (2omf.pdb). What type of fold is bacterial porin? The complete trimer can be seen in 2omf_mmol.pdb. 2. Can you determine how the protein interacts with its environment? Is it amphiphilic? If so how? Make use of the Java implementation of the hydrophobicity plot Plots.htm simply paste in the amino acid sequence. The amino acid sequence of the porin is supplied in file 2omf_fasta.aa. The core of the bacterial outer membrane is about 25Å. 3. How does porin regulate passage of solute molecules? What is the role of the long loop between strands 5 and 6? Exercise 3 K + channel The bacterial K + channel is a functional tetramer made of identical subunits. Unlike porin, the fold of the K + channel uses a motif of three α helices. 1. Take a look at one subunit of the tetramer (1bl8.pdb) and examine the three α helices that make up the motif. RasMol> restrict *A Are the ions represented in the coordinate file? If so, be sure to display them. 2. Can you determine how the K + channel protein interacts with its environment? Is it amphiphilic? If so how? Use the Java program as above. The amino acid sequence is provided (1bl8_fasta.aa). 3. What is the structural basis of specificity in the K + channel? The K + ion (1.35 Å radius) is favored over Na + (0.95 Å radius) by a factor of 10,000, even though Na + is smaller. So it cannot simply be a steric effect. Look for the selectivity filter (Brandén & Tooze p ) in the molecule. 4. Compare and contrast globular proteins and membrane proteins in terms of 1) distribution and content of amino acids, 2) major determinants of structure, and 3) representation in the structural databases.

5 Answer sheet Part 1 Catalysis Exercise 1 Trypsin Mechanism of base-catalyzed amide hydrolysis What type of structures (fold) are they? Notice how the fold brings these sequentially distant active site residues together. What do the terms specific and non-specific mean in terms of Branden & Tooze s description? The peptide bond is cleaved only very slowly (weeks!) in this inhibitor by the enzyme. Why should this be different than more general substrates? Explain (using transition state theory) how enzymes function. Be sure to make diagrams to support your explanation. Explain (using transition state theory) two possible ways in which inhibitors may function. Be sure to make diagrams to support your explanation.

6 How do the preformed structures affect the thermodynamics of complex formation? Try to identify this oxyanion hole in the 3D structure of trypsin. Does the structure also provide the means for decomposing the acyl-enzyme intermediate? Or is it left to chance? Explain. Exercise 2 Subtilisn What type of structure fold is this inhibitor? Do either of the enzyme or inhibitor have SS bonds? What is the handedness (topology) of the unusual β α β motif? Find those residues on Subtilisn responsible for this specificity. Notice the interaction between the inhibitor residues and Subtilisn residues and What kind of interaction is formed? Do solvent water molecules play a role in recognition? Catalysis? Part 2 Membrane proteins Exercise 1 Bacteriorhodopsin Take a look at bacteriorhodopsin (1ap9.pdb). Identify the 7 helices and the retinal ligand. Can you determine how the protein bacteriorodopsin interacts with its environment? Is it amphiphilic? If so how? Make use of the Java implementation of the hydrophobicity plot.

7 Exercise 2 Porin What type of fold is bacterial porin? Can you determine how the protein porin interacts with its environment? Is it amphiphilic? If so how? Make use of the Java implementation of the hydrophobicity plot. How does porin regulate passage of solute molecules? What is the role of the long loop between strands 5 and 6? Exercise 3 K + channel Can you determine how the K + channel protein interacts with its environment? Is it amphiphilic? If so, how? What is the structural basis of specificity in the K + channel? Compare and contrast globular proteins and membrane proteins in terms of 1) distribution and content of amino acids, 2) major determinants of structure, and 3) representation in the structural databases.