Enzymes Essays Flashcards
What are the modules of interest in cellulases
Catalytic modules, cellulose binding modules, linkers, cohesins and dockerin modules
What the are modules of interest in aminoacyl tRNA synthetases?
Direct catalytic modules, extra indirect catalytic modules, modules for extra amino acids conversion, tRNA recognition, editing domains, MSC and extra functions.
What are the benefits to SBDD?
Save money, use homologs, able to choose target
What are the limits of SBDD?
Must know structure, doesn’t predict effect on system, doesn’t identify which ligands to take forward. And must know which structure you want to target.
What are the topics of the SP and ACE essay?
Mechanism of action, optimisation for substrates, inhibition, accessibility, convergent and divergent evolution.
What do cellulases do?
Hydrolyse the 1,4-beta-D-glycosidic linkages in cellulose
What are the different catalytic modules of cellulases?
Cellobiohydrolase (CBH I and II), Endoglucanase, Beta-glucosidase
What does CBH I do?
Hydrolyse the reducing end of cellulose
What do endoglucanases do?
Cleave in less ordered regions to expose more ends
What do beta-glucosidases do?
Cleave free beta-glucosidases
What is the properties of cellulose?
Highly ordered crystalline repeats of beta-glucose, with intermittent regions with less order.
What is the benefit to many catalytic modules?
Can target complex substrate from more angles = more yield.
How does the catalytic binding module of cellulases bind?
Binds with loose, transient bonds using exposed aromatics spread one glucose module apart.
Why are catalytic binding modules of cellulase helpful?
Insoluble substrate less likely to collide with correct orientation (thread through CBH I correctly)
How does catalytic binding module of cellulase help?
Disrupts crystalline structure and brings CMs closer
What is the linker of cellulases used for?
Flexibly joins CM to other modules, aiding positioning
Give an example of a cellulase linker
Fibronectin type III
What is the structure of cohesins and dockerins?
Highly conserved and species specific
What is the role of cohesins and dockerins?
Tether modules together, increasing proximity and activity, allows inclusion of non-cellulases. SYNEGRY
What does cellulases bind to with their dockerin modules?
Scaffoldins, which can bind to other scaffoldins.
What is the class I for direct aminoacylation domain?
2’ carbon, which moves by transesterification. Conserved Rossman fold, with HIGH and KMSKS motifs.
What is the class II for direct aminoacylation domain?
3’ carbon. Less conserved, unrelated beta-sheets.
Why are there many catalytic domains of aaRSs
Because amino acids structure varies.
What is indirect charging of tRNA?
Charging incorrect AA to tRNA, which is then transamidated by amidotransferase to give the correct AA.
When is indirect charging of tRNA needed?
Aspartic and Glutamic acid are converted to Asparagine and Glutamine.
Why have some organisms got more domains for charging?
Expand from the naturally occurring AAs: formyl-methionine, selenocysteine and pyrrolysine. Converts from methionine, serine and lysine.
Which domains recognise tRNA?
Acceptor stem binding domain and Anti codon binding domain.
Why is tRNA recognition important?
Differentiate between the 64 codons, and anticodon binding domain provides stability for transport.
What do the editing domains do?
Kinetic proof reading, pre or post acylation (in solution or selective release).
When are editing domains needed?
Only for amino acids which regularly get mischarged: Ile, Leu and Val. They are a defence mechanism.
What is the purpose of a multisynthetase complex?
Promotes activity, channelling product to ribosomes. Localise, stabilise and act as a reservoir to regulate non-canonical activities.
What extra functions are aaRS responsible for?
Metabolism, angiogenesis, and Immune functions.
What example of SBDD is used for saving money?
Cellulase not same as AChE (active site and gorge)
What example of SBDD is used for needing to know the structure?
Lysozyme easy to crystallise
ACE not easy
Why is ACE not easy to crystallise?
Glycosylated and membrane bound
What example of SBDD is used for use of homologs?
ACE and carboxypeptidase A, because both metalloproteases. But ACE turned out to be more similar to neurolysin.
What assumption of ACE active site was made?
Zn, H bonding region, and positive residue.
Using homologs allowed what to be developed?
Captopril, Elanapril, and Lisinopril.
What was good and bad about using homologs for ACE inhibitors.
Allowed life saving drugs to be developed cheaply. But had side effects like dry cough and sore throat.
What example of SBDD is used for knowing which drug to screen?
LBDD: Hirudin from leeches to Bivalirudin, and Brazillian snake venom to captopril.
Why is knowing which drug to screen a problem is SBDD?
Knowing pharmacophores doesnt mean you know which potential inhibitors will be a good picture.
What is Bivalirudin?
A thrombin inhibitor, doesnt target other serine proteases. Binds reversibly to free and clot-bound thrombin.
What example of SBDD is used for “predicting the effect on the system”?
Glycosylation by golgi alpha-mannosidase
How can glycosylation pathways be imbalanced?
Too much alpha-mannosidase activity, too many complex sugars = cancer.
Too little, too many hybrid = lupus-like autoimmunity.
Why is having to choose the correct structure a limitation to SBDD?
Many proteins are present in systems, choosing the right one is hard.
Why is having to choose the correct target a benefit to SBDD?
Unlike LBDD, you can target domains specific to certain isoforms
Give two examples of choosing the right target in SBDD.
COX enzymes and ACE enzymes.
Why is COX enzyme an example of SBDD “choosing the right target”?
COX turn arachidonic acid to prostanoids (for inflammation and pain). COX1 low levels in GI, NSAIDS. COX2, extra domain, only in cellular stress, Vioxx and celecoxib.
Why is ACE enzyme an example of SBDD “choosing the right target”?
Somatic (2 domain) and Germinal (C domain).
C = cleaves angio, bp control. No increase in bradykinin causing an angioedema.
N = Cleave AcSDKP and haemoregulatory hormone.
Why are Serine Proteases comparable to AChE?
Same catalytic triad (Ser, His, Carboxy acid - Glu) for hydrolysis.
What is the mechanism for SP/AChE cleavage?
G+H low barrier H bond, raise pKa and basicity, attacks S
S O- attacks substrate = tetrahedral oxyanion.
H proton attacked, form acyl intermediate, release product.
Repeat with H2O, release second product.
How have SPs optimised for their substrates?
Binding pocket for n-1 residue
Trypsin = positive residue
Elastase = small neutral residue
Give an example of SP specificity
Thrombin cleaves proteins such as fibrinogen for blood coagulation.
How is inhibition of SP regulated? Why?
Serpins, prevent protease activity in unwanted areas.
How do serpins work?
A prime hydrolysis target, the reactive centre loop, has tension. Moves to relaxed state once acyl-intermediate is formed, preventing its release.
Why is AChE active site not easily accessible?
Its at the bottom of a 20A gorge; lined with 14 aromatic amino acids.
How is AChEs lack of accessibility overcome?
Proposed back door mechanism, releasing products. Kineticist say it isn’t needed.
How is type of evolution assessed?
Sequence identity
What examples of convergent evolution are there?
AChE with gorge against chymotrypsin-like SPs against subtilisin-like SPs
What is the difference in serine proteases structure?
Chymotrypsin-like = 2 beta-barrels Subtilisin-like = large beta-sheet with several alpha-helices
What examples of divergent evolution?
Amongst chymotrypsin-like serine proteases, a high sequence identity. Difference in the binding pocket.
Also cysteine proteases may have diverged.
