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MBB261 > Rice > Flashcards

Rice Flashcards

1
Q

Why study enzymes?

A
  • pose challenging and interesting questions
  • excellent catalysts even though limited repertoire of functional groups
  • operate in water at 37° and pH4-9 (also extremophiles)
  • often use add chem species, eg. metals, coenzymes, to augment functions of 20AAs
  • medium sized molecules –> are parts of protein removed from active site important?
  • key to metabolism so hope to soon predict function from gene seq and understand control and reg of metabolism
  • inhibitor design to modulate metabolism
  • goal of designing new enzymes de novo
  • industrial use of enzymes big business
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2
Q

Where are enzyme names usually derived from?

A
  • substrate or reaction catalysed and ase
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3
Q

What are isoenzymes?

A
  • same function and same basic name

- but diff AA seqs

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4
Q

How can isoenzymes sometimes be distinguished?

A
  • diffs in optimal pH, kinetic properties or immunology
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5
Q

What is the nomenclature for enzymes?

A
  • described by 4 no.s
  • 1st broadly classifies mechanism
  • then subdivided into sub-families
  • then sub-sub-classes
  • 4th no. specific to enzyme
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6
Q

What are the 6 broad classifications of enzyme?

A
  • oxidoreductases = cat ox/red reactions
  • transferases = transfer functional group
  • hydrolases = cat hydrolysis of various bonds
  • lyases = cleave various bonds, not using hydrolysis/ox/red
  • isomerases = cat intramol isomerisation changes
  • ligases = join 2 molecules by covalent bonds
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7
Q

What was the lock and key model?

A
  • substrate exact fit for active site and forms ES comples
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8
Q

What was Haldene’s enzyme model?

A
  • substrate in shape complementary to transition state, as stabilisation enhances rate
  • most of enzyme has no active role and req for optimal orientation of active site
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9
Q

What was Pauling’s model?

A
  • substrate complementary to active site

- strong bonds to transition state and weak to reactants/products

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10
Q

What was Koshland’s model?

A
  • induced fit
  • enzymes flexible and active site continually reshapes as result of interactions w/ substrate until completely bound, then catalysis occurs
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11
Q

What is the transition state?

A
  • transient entity formed in conversion of reactants to products
  • max energy point along reaction path
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12
Q

Can the transition state be isolated, why?

A
  • no
  • lifetime is 1 bond vibration
  • may have partial or imaginary bonds and more ligands bound to atom than standard valency supports
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13
Q

What is activation energy?

A
  • diff between energy of transition state and reactants
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14
Q

What does the energy profile diagram for a 1-step chemical reaction show?

A
  • DIAG*
  • larger the activation energy, the slower the reaction
  • larger the net free energy charge, the more irreversible the reaction
  • exo reaction has products of lower free energy than reactants
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15
Q

What does the energy profile diagram for a 2-step chemical reaction show?

A
  • DIAG*
  • intermediate formed in conversion of reactants to products
  • transition state remains point of highest energy
  • activation energy still diff between energy of transition state and reactants
  • can only isolate intermediate if energy low enough
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16
Q

What does Pauling’s concept as an energy profile diagram show?

A
  • DIAG*

- enzyme catalysis lower energy of transition state by more than lowering of reactants/products

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17
Q

What are the diff types of catalysis enzymes use?

A
  • covalent –> nucleophilic and electrophilic
  • acid
  • base
  • metal ion –> electrophilic, ligand activation, redox
  • strain
  • conformational organisational –> entropic control
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18
Q

What are some of the key functional groups in enzymes?

A
  • Cys, Ser/Thr, Tyr, Asp/Glu, Lys, His, Asn
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19
Q

What is the role of co-enzymes in enzyme catalysis?

A
  • augment role of key AAs
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20
Q

What are the properties of the C-O-H group in Ser/Thr?

A
  • DIAG*

- no resonance structures for anion, so not acidic (pKa = 15)

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21
Q

What are the properties of the C-O-H group in Tyr?

A
  • DIAG*

- 4 resonance structures of phenolate ion, so mod acidic (pKa = 10)

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22
Q

What are the properties of the C-O-H group in Asp/Glu?

A
  • DIAG*

- 2 resonance structures for carboxylate anion, so quite acidic

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23
Q

What are the properties of the C-N-H group in Lys?

A
  • DIAG*

- no resonance structures, so weak acid and strong base (pKa ≈10)

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24
Q

What are the properties of the C-N-H group in His?

A
  • DIAG*

- imidazole cation stabilised by 2 equivalent resonance structures, so moderate acid and base (pKa ≈7)

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25
Q

What are the properties of the C-N-H group in Asp/Gln?

A
  • DIAG*

- carboxamide has 2 resonance structures and no lone pair on nitrogen, not basic

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26
Q

What do Ser proteases do?

A
  • cleave polypeptides
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27
Q

How does chymotrypsin acts a Ser protease?

A
  • uses Ch2OH of Ser as nucleophilic covalent catalyst to form acyl-enzyme intermediate
  • uses His-57 as proton donor/acceptor
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28
Q

Are there diff classes of proteases, give examples?

A
  • several major classes, defined by active site residue

- eg. serine proteases, thiol proteases, metallo proteases

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29
Q

What are some of the functions of Ser proteases?

A
  • many diff

- eg. protein digestion, blood clotting

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30
Q

How are substrate specificity pockets of proteases defined?

A
  • pockets S1, S2 etc. upstream of scissile bond

- pockets S1’, S2’ etc. downstream of scissile bond

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31
Q

What is the role of specificity pocket S1?

A
  • binds residue p1, the main specificity site after which cleavage occurs
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32
Q

Why do proteases have 1 or more substrate specificity pockets?

A
  • can select for certain types of side chain
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33
Q

How are some proteases much more specific?

A
  • may recognise side chains in other specificity sites, or may recognise substrates main chain conformation using their other specificity sites
  • eg. thrombin has 2 pockets, like hand (side chain) and glove (pocket)
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34
Q

How was chymotrypsin initially synthesised?

A
  • as inactive enzyme precursor called chymotrypsinogen
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35
Q

Where does chymotrypsin cleave?

A
  • cleaves polypeptides after large aromatic residues (Phe, Tyr, Trp)
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36
Q

What key residues did chem mod studies of chymotrypsin identify?

A
  • essential Ser195

- essential His57

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37
Q

How can Ser195 of chymotrypsin be specifically mod?

A
  • PMSF –> used in preps to block Ser proteases, total inhibition DIAG
  • DIPF –> blocks Ser proteases and related molecules, eg. acetylcholinesterase involved in synaptic transmission in CNS DIAG
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38
Q

How can active site His57 of chymotrypsin be chemically labelled?

A
  • TPCK is substrate analogue w/ reactive groups, binds at active site of enzyme and reacts w/ His
  • 1:1 of enzyme-TPCK complex formed
  • His57 mod, so enzyme inactivated
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39
Q

What is the structure of chymotrypsins polypeptide chain?

A
  • folds into 2 β-barrels, each formed from 6 anti-parallel β-strands
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40
Q

What makes up the catalytic triad of chymotrypsin and where are they found?

A
  • His57, Ser195 and Asp102

- found in deep cleft

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41
Q

Apart from the catalytic triad, what are the other key features of chymotrypsin?

A
  • oxyanion hole –> res 193-195
  • specificity pocket –> res 189, 216, 226
  • main chain –> res 214-216
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42
Q

How are 3 residues of catalytic triad held in exactly right orientation?

A
  • adj residues conserved to hold them

- after this bit more flexibility, so semi conserved

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43
Q

How does the catalytic triad function?

A
  • interaction makes it much easier to stabilise -ve charge on Ser195
  • -ve Asp102 stabilises formation of +ve form of His157, helping His57 grab Ser195 proton
  • makes Ser195 nucleophilic, as highly reactive against substrates or inhibitors w/ δ+ charge
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44
Q

Where is the oxyanion hole and what is its role?

A
  • located near carbonyl group of substrates scissile bond
  • name denotes region in active site where backbone amide hydrogens of Ser195 and Gly193 point into active site cavity
  • these amino groups positioned so tetrahedral enzyme-substrate intermediate stabilised
  • increases enzyme activity 10,000x
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45
Q

What is the mechanism for chymotrypsin?

A
  • His57 acts as base and pulls proton off Ser195, so Ser transiently O- and can atack
  • Asp102 also assists charge relay
  • powerful nucleophile Ser195 attacks unreactive substrate carbonyl
  • enzyme briefly covalently bonded to substrate, forming tetrahedral intermediate, -ve charge stabilised by oxyanion hole
  • His57 now acts as acid and donates proton
  • decomposes to acyl-enz intermediate
  • water attacks nucleophile and Ser195 is leaving group
  • deacetylates enzyme by reversing decomposition through another tetrahedral intermediate
  • enzyme returns to original state
  • release of polypeptide w/ new C-ter
46
Q

What is the chymotrypsin mechanism an example of?

A
  • coordinated operation of nucleophilic covalent catalysis and general acid-base catalysis
47
Q

What is the general overall reaction for the mechanism of chymotrypsin?

A
  • DIAG*

- enz + substrate enz-substrate complex en-prod 2 complex + prod 1 enz + prod 2

48
Q

What are the similarities and diffs between trypsin, chymotrypsin and elastase (all Ser proteases)

A
  • similar seqs and structures

- diff specificities

49
Q

Where do diff Ser proteases cleave their polypeptide substrates?

A
  • chymotrypsin = after large aromatic side chains (Phe/Tyr/Trp)
  • trypsin = after long +ve AAs (Lys/Arg)
  • elastase = after small hydrophobic AAs (Ala/Val/Thr)
50
Q

How similar are the seqs of trypsin to chymotrypsin and elastase?

A
  • share ≈40% seq identity
  • only divergence in specificity
  • mechanism identical
51
Q

How do the specificity pockets of trypsin, chymotrypsin and elastase vary, and why?

A
  • chymotrypsin has Ser as don’t want -ve charge
  • trypsin has Asp to attract +ve AAs and deep pocket for long AAs
  • elastase has AAs in side to block off parts so can recognise smaller side chains
52
Q

How do trypsin, chymotrypsin and elastase recognise the substrate backbone, and why is it important?

A
  • recognise its conformation
  • helps orientate polypeptide for cleavage
  • anti-parallel β-sheet formed between substrate and protein
53
Q

How is thrombin more specific?

A
  • more elaborate side chain and main chain recognition sites, so limits no. substrates they can bind and cleave
54
Q

Why do enzymes like chymotrypsin need to be carefully controlled?

A
  • destroy other proteins, so could digest own intestines
55
Q

How are enzymes like chymotrypsin controlled?

A
  • prod as inactive zymogens
  • need proteolytic cleavage (irreversible) of precursors to activate them
  • ,aster activator of protease activation cascade is enterpeptidase
56
Q

How is chymotrypsinogen converted to active chymotrypsin?

A
  • DIAG*
  • partly activated by trypsin –age> this 1st cleavage prod new NH3+ group on res 16
  • prod π-chymotrypsinogen, which is able to activate other π-chymotrypsinogen molecules
  • prod fully active α-chymotrypsinogen
  • 3 parts remain connected by disulphide bonds throughout
57
Q

How is the active site of chymotrypsin activated?

A
  • in chymotrypsinogen neither catalytic triad or oxyanion hole are fully formed (so inactive)
  • on cleavage by trypsin new N-ter NH3+ group on Ile116 pairs to side chain of Asp194 – > alt main chain conformations between residue 193-195
  • changes position of Ser195 side chain, forming correct geometry for catalytic triad
  • also orientates main chain amides of res 193 and 195 –> forming oxyanion hole
  • enzyme now active
58
Q

How is the blood clotting activation cascade highly controlled?

A
  • key step is thrombin cleaving Arg-Gly bond in soluble protein fibrinogen
  • elaborate activation and deactivation system based on Ser proteases w/ +ve and -ve feedback loops
  • 2nd system based on plasmin/plasminogen for dissolving clots
59
Q

What is another similar Ser protease based cascade to blood clotting activation cascade?

A
  • control of complement system in immune response
60
Q

What is a classic example of convergent evo at the mol level?

A
  • bacterial protease subtilisin and chymotrypsin
  • completely unrelated seq and structure
  • but evolved to converge on same solution to carry out proteolysis reaction –> also has catalytic triad involving Asp, His and Ser
61
Q

What are the 2 types of acid-base catalysis?

A
  • specific acid-base catalysis

- general acid-base catalysis (GABK)

62
Q

What is specific acid-base catalysis, and when is it used?

A
  • proton fully transferred before covalent bonds made/broken

- what chemists mainly use

63
Q

What is general acid-base catalysis, and when is it used?

A
  • proton transferred at same time as covalent bonds made. broken
  • what enzymes mainly use
64
Q

Why is general acid-base catalysis called general?

A
  • all acids in mixture contribute as proton donors in proportion to acid strength
  • any generalised base can act as proton abstractor (typical AA side chains used are His, Asp, Glu, Tyr, Lys
65
Q

Why is it called acid-base catalysis?

A
  • as AAs in forward reaction also act as bases in reverse reaction
66
Q

What is the structure of 20S proteasomes, and how do they function?

A
  • intracellular
  • multi-subunit
  • cylinder shaped complexes
  • interior cave containing proteolytically active sites mechanistically belonging to N-ter Thr hydrolases
  • Thr at N-ter of β-subunit acts as attacking nucleophile assisted by sidechain of neighbouring Lys and N-ter amino group
67
Q

Why are inhibitors targeting proteasome being researched?

A
  • important target for drug dev
68
Q

What is the mechanism for carboxypeptidase?

A
  • metal ion catalysis
  • zinc ion at active site tetrahedrally coord by 2 His and a Glu
  • water activated as attacking nucleophile, by zinc ion, w/ assistance of Glu270 which pulls proton off
  • so water is 4th ligand, directed to amide carbonyl and O- attacks C
69
Q

Why is there no covalent intermediate between the enzyme and substrate in the reaction mechanism of carboxypeptidase?

A
  • as water is the attacking nucleophile
70
Q

What are aspartyl proteases?

A
  • a further class of proteolytic enzymes which inc highly substrate selective HIV protease
71
Q

Why is HIV protease a target for drug discovery?

A
  • due to rate of virus maturation
72
Q

How is the structure/mechanism-based drug discovery targeting HIV?

A
  • in HIV protease active site, 2 Asp clamp water molecule which acts at attacking nucleophile
  • key element in drug affinity is 2° alcohol that mimics transitions state of reaction
73
Q

What is the mechanism of the Cys protease, Papain?

A
  • uses thiol group of Cys25 and imidazole of His159 to form ion pair –> poss due to low pKa of Cys (8) compared to Ser (≈16)
  • so enzyme had nucleophilic thiolate anion which attacks peptide bond
  • acyl intermediate formed following release of 1st product is moderately stable
  • in 2nd half of reaction water is nucleophile to hydrolyse thiol ester and form carboxylate as 2nd product
74
Q

How does the reaction mechanism of Papain differ from Ser proteases?

A
  • in Ser protease O-H bond broken as bond between O and substrate formed
  • compared to Papain where nucleophilic thiolate anion attacks peptide bond
75
Q

What are the types of protease and what is their key characteristic?

A
  • Ser protease –> Asp/Glu, His and Ser triad
  • Thr protease –> use hydroxyl of Thr as nucleophile
  • Cys proteases –> His-Cys diad
  • Aspartyl (acid) proteases –> use 2 carboxyl groups to activate water
  • metalloproteases –> use metal ion to activate water
  • eqolysin –> uses Glu to activate a water
76
Q

What do all types of protease have in common?

A
  • nucleophile (Ser/Thr/Cys/water) activated by another part of active site (His/Asp/Zn2+ etc.)
  • other groups on enzyme polarise carbonyl group of peptide to be cleaved
  • -vely charged tetrahedral intermediate prod, stabilised by enzyme (eg. oxyanion hole in chymotrypsin)
  • intermediate collapses to prod cleaved peptide
77
Q

Why is Burkholderia an emerging threat to health?

A
  • multi-drug resistant strains
  • currently no vaccine
  • biology complex and poorly understand
  • 7 morphologies w/ diff patterns of gene expression can be identified
78
Q

What has proteomic analysis of pathogenic and non-pathogenic Burkholderia strains shown?

A
  • 14 hypothetical proteins of unknown function
  • BPSL1549 is 23kDa protein of unknown function and unremarkable seq showing no seq similarity to any protein outside Burkholderia
79
Q

What did fold comparisons show BPSL1549 had a remote relationship w/ in a diff protein?

A
  • showed structural, but not seq similarity w/ catalytic domain of CNF1
  • spatial conservation of His-Cys pair, w/ orientation reminiscent of catalytic pair in Papain, which hydrolyse peptide bonds
80
Q

What is CNF1?

A
  • flesh eating factor

- 114kDa toxin expressed by some pathogenic E. coli strains

81
Q

What is the role of CNF1?

A
  • deamidates key Glu in family of small GTPases Rho, Rac and Cdc42
  • blocks GAP-dep deactivation of GTPase by inhibiting GTP hydrolysis
  • leads to remodelling of actin cytoskeleton DIAG
82
Q

What does catalysis in CNF1 involve?

A
  • key Cys, spatially conserved in BPSL1549, mutation of which to Ser abolishes all enzymatic activity of toxin
83
Q

Is cleavage of an ester bond easier or harder than cleavage of a peptide bond?

A
  • slightly easier
84
Q

What do lipases do?

A
  • catalyse conversion of triglycerides to monoglycerides and free fatty acids
85
Q

What applications do lipase inhibitors have?

A
  • controlling obesity
86
Q

How do lipases hydrolyse triglycerides?

A
  • sequentially cleave 1 chain at a time at ester bond
  • triglyceride –> diacylglycerol –> monoacylglycerol
  • in each reaction water converted to RO2
  • DIAG*
87
Q

What does pancreatic lipase hydrolyse triacylglycerol to?

A
  • 2-acylglycerol
88
Q

What is the function of colipase in the complex formed between human pancreatic lipase and pig colipase, and where does the alkyl phosphonate inhibitor bind?

A
  • catalytic triad present at active site
  • colipase important protein cofactor that promotes hydrolytic activity of lipase subunit β-strands, helices and loops
  • inhibitor binds at side of catalytic triad
89
Q

What is the mechanism of lipase?

A
  • His removes proton from Ser in catalytic triad once substrate has bound
  • O acts as nucleophile to attack C and push e-s out of O to double bond, when e-s come back can remove leaving group
  • O- goes onto O of carbonyl
  • oxyanion hole formed to stabilise O-, by H bonding to main chain of other parts of lipase
  • after alcohol left, acyl enzyme intermediate resolved by water entering active site
  • products resolved (Ser and acid)
90
Q

How does lipase mechanism differ to that of Ser proteases?

A
  • same, except leaving group is O- instead of N-
91
Q

Why study lipases and give an example?

A
  • clear medical apps

- eg. tetrahydrolipstatin is a potent phospholipase inhibitor used to treat obesity

92
Q

How is pancreatic lipase inhibited by lipstatin inhibitors?

A
  • DIAG*
  • active covered by lid like domain
  • interaction w/ bile salt micelles and colipase makes active site available
  • β-lactone ring of lipstatin/tetralipstatin inhibitor interacts w/ Ser at active site, inhibiting the enzyme
93
Q

Why do bacteria secrete a range of toxins?

A
  • harm other bacteria to colonise env

- sometimes to colonise people, causing disease

94
Q

What does the bacterial type VI secretion system consist of?

A
  • approx 13 polypeptides that form molecular machine of 100s of protein subunits
  • sheath tube machinery that sits in IM and sequesters molecules to be ejected from cell
95
Q

What does the bacterial type VI secretion system do, and how?

A
  • needle like device goes from state sat in inner cellular space to needle being thrusted through OM
  • where interacts w/ other organisms and secrete proteins into extracellular space or into other bacteria
96
Q

What are needle like machinery in bacterial type VI systems related to in 3D structure?

A
  • structures bacteriophages use to inject other bacteria
97
Q

Who does the Burkholderia cenocepacia pathogen infect?

A
  • CF patients
98
Q

How does Burkholderia cenocepacia secrete toxins and what is 1 of them?

A
  • using type VI secretion system

- 1 is a lipase

99
Q

How does Burkholderia cenocepacia protect itself from the toxins it produces?

A
  • makes immunity proteins which inactivates lipase by trapping lid, preventing it closing on active site, to gen active structure of enzyme
100
Q

What family is mandelate racemase a member of?

A
  • enolase superfamily
101
Q

What reaction does mandelate racemase catalyse?

A
  • interconversion of R-mandelate and S-mandelate
102
Q

How does mandelate racemase catalyse this reaction?

A
  • inversion of chiral centre to remove proton and add it to other side
  • DIAG* - if remove H from this molecule then v high energy (not stable) and can’t push -ve charge anywhere useful
  • DIAG* - now can delocalise -ve charge over carboxyl, giving double -ve charge
  • DIAG* - aci-carboxyl intermediate higher energy than carboxyl, but could stabilise and favour formation by having metal ion bound (+ve charge), then can pick up proton had at start and go back to beginning of reaction, or if proton donor on other side can pick it up and prod other enantiomer
103
Q

What do the enolase superfamily have in common?

A
  • cluster of residues involved in binding metal ion (stabilises aci-carboxylate)
  • use enzyme bound Mg ion to facilitate catalysis
  • capping domain which provides residues that determine nature of chemical enzyme can bind
  • residues at interface of capping domain and barrel domain provide substrate binding specificity
  • residues involved in acid-base catalysis in barrel domain
104
Q

What are the diffs between enzymes of enolase superfamily?

A
  • after remove proton from C α to carboxyl group and gen intermediate, then charge comes back in, and what happens next dep on enzyme
  • residues in barrel domain (involved in acid-base catalysis) differ as dictated by chemistry
105
Q

What is the mechanism of mandelate racemase mechanism?

A
  • metal ion near carboxylate, pull of proton, gen carbanion which can be stabilised by formation of aci-carboxylate intermediate
  • uses Lys as base to remove L-proton and His as acid to return D-proton
  • can go either way
106
Q

Do R and S-mandelate have the same energy, and what is the importance of this?

A
  • yes

- so eq established as 50:50 mixture

107
Q

What are the diff consequences of removal of roton α to carboxylate in enolase, muconate lactonising enzyme (MLE) and mandelate racemase (MR)? Show structure of substrate, intermediate and product?

A

DIAG x9

108
Q

What do the reactions of the substrate to form the intermediate have in common in enolase, MLE and MR?

A
  • CHO2, removed to make aci-carboxylate

- shift charge to O by pulling off proton

109
Q

What is the structural similarity of enolase, MLE and MR?

A
  • overall level seq identity v low (<3%) but has other conservative subs
  • similarity closest around binding site for Mg ions
  • other residues that dictate some of catalytic properties have to change as chem isn’t exactly the same (closely related intermediate but enzyme does something diff)
  • but structures v similar and have same fold
  • conservation highest in active site and esp w/ residues that bind metal ion
110
Q

Is methylaspartate ammonia lyase similar to the enolase superfamily?

A
  • exactly like them in structure
111
Q

What does methylaspartate ammonia lyase do?

A
  • converts methylaspartate to mesaconate through loss of NH3
112
Q

What is the mechanism of methylaspartate ammonia lyase?

A
  • side chain of Lys331 acts as base to remove proton α to carboxyl group
  • gen enolic aci-carboxylate intermediate, stabilised by Mg ion
  • subsequent collapse of aci-carboxylate shifts double bond and leads to elimination of amino group to yield mesaconic acid and NH3

MBB261 (6 decks)

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