From the PreAssignment
Use of the enzyme nomenclature database.

a.     Goal I - Use the Enzyme nomenclature database to find the common names of the two proteins closest to TOP.

                                               i.     Go to http://www.expasy.org/

                                             ii.     Resources A..Z, Find ENZYME in list (You can also find it under Categories: Proteomics) 

                                            iii.     Click – by enzyme class

                                            iv.     Analyze the naming system

1.     What type of reaction do enzymes in the E.C 1 ._._._  catalyze? (Know 1-6 and definitely 1 and 3)

2.     What type of reaction do enzymes with the E.C. 5.2._._ catalyze. What specific substrates are acted on?

                                              v.     Find E.C. 3.4.24._

1.     What type of reaction do enzymes of this class catalyze?

2.     What does the 4 stand for?

3.     What does the 24 stand for?

                                            vi.     Click on 3.3.24._

1.     What are the common names of the two enzymes closest to EC 3.4.24.15?

b.     Goal II – Find the EC numbers for thermolysin and hexokinase. What reactions do the enzymes catalyze?

 

 

2.     Sequence analysis

a.     Now lets see if an enzyme can be identified with significant sequence similarity to EC 3.4.24.15 by running a BLASTp

                                               i.     Go to http://www.expasy.org/

                                             ii.     Click on Categories: Proteomics (left  column)

                                            iii.     Find BLAST

                                            iv.     Scroll down to the different types of BLAST searches. What is the practical difference between  tBLASTn and BLASTp. Under the two BLASTp boxes investigate the various databases that you have access to from the two drop down menus.

                                              v.     Enter the accession number for Human E.C 3.4.24.15

P52888 (can you find this on EXPASy?) 

                                            vi.     Scroll down and make sure first Blastp is selected

                                            vii.     Notice you can select various databases for the search. Select Eukaryota

                                           viii.     Check the box - Search only Swiss-Prot (curated sequences)

                                            ix.     Scroll to the bottom of the page. Set E threshhold to 1. Run Blast

                                             x.     What are the common names and EC numbers for the two enzymes with the most sequence similarity to Thimet Oligopeptidase? (There should be lots of both listed)

                                            xi.     Scroll down to observe the sequence alignments

                                          xii.     What is the % identity between human TOP and human Neurolysin? Are the two proteins likely to have similar structures? Explain?

 

 

b.     Lets look for important residues based on sequence similarity by comparing multiple sequences using ClustalW2

                                               i.     Put check marks next to P52888, P24155, Q9BYT8, P25375, Q99797

1.     What are the names and EC numbers for these enzymes?

                                             ii.     Click Submit Query (Next to Clustal W (multiple sequence alignment)

                                            iii.     (A new page should open up. Notice it has submitted your selected list of protein sequences in FASTA format). Click Run ClustalW (You can enter your email but I find it more convienient to have the results appear in the browser window)

                                            iv.     On the next page click clustalw (aln) to view your results 

1.     What do the symbols . : and * signify?

2.     Suppose you suspect that Tyr612 of the human TOP enzyme may be significant for enzyme activity. Find Tyrosine 612 of THOP_Human. Are you suspicions confirmed or in doubt? Explain?

 

3.     Structure Comparison

a.     We predicted based on sequence that TOP (EC 3..4.24.15) and Neurolysin (EC 3.4.24.16) are likely to have similar structures. Lets see if this is the case.

                                               i.     Go to the EMBL-EBI wesbite and follow the links to the SSM (Secondary Structure Matching) tool

Home Databases PDBe Services PDBeFold

                                             ii.     Read the information about SSM

                                            iii.     Click the Start SSM button

                                            iv.     Enter 1S4B (pdb code for the structure of human TOP)

                                              v.     Make source is set to All PDB archive

                                            vi.     Set lowest match to 50%

                                           vii.     Click the Submit Your Query Button

                                         viii.     Select 1i1i (Neurolysin) by clicking on the number

                                            ix.     Click View Superposed

1.     How well matched are the structures? Where do the two enzyme structures differ the most?

b.     Lets overlay the structure using the Combinatorial Extension (CE) Method

                                               i.     Go to http://cl.sdsc.edu/ (CE link on our website)

                                             ii.     Click Two Chains

                                            iii.     Enter the PDB codes for TOP and Neurolysin

                                            iv.     Click the Calculate Alignment Button

                                              v.     What are the RMSD and sequence similarity of the proteins?

                                            vi.     Scroll down and compare the 3D structures