6:Structure-Function Relationship Flashcards
(36 cards)
Proteases
enzymes that cleave peptide bonds in proteins
Kinases
enzymes that catalyse the phosphotransfer from ATP to protein side chains
Enzyme summary 4+1
catt. chem reactions by changing kinetics (rate of reaction) NOT td.
higher reaction rates
high specificity for substrates
permit regulation by cellular signalling
> stablise TS, lower Ea, inc reaction rate
what specificities can enzymes have 2
geometric specificity for substrates
stereochemical specificy
types of catalysis: Acid-Base Catalysis 2
lowers TS energy by partial proton transfer
usually with histine
types of catalysis: covalent catalysis 2
transient formation of enzyme-substrate cov. bond
can have nuc/electrophillic steps
types of catalysis: metal ion cat.
metalloenzymes vs metal-activated enzymes
role of ions 3
1/3 of enzymes req metal ions
metalloenzymes vs metal-activiated enzymes
metallo = tightly bound
metal activated = loosly bound and easily exchanged
ions act via:
binding/orientating substrates
mediating redox
electrostatically shielding or stabilising -‘ive charges
types of catalysis: electrostatic cat.
1+3
gen electrostatic potential around enzyme/in active site
> dielectric constant in active site (WITHOUT WATER) is closer to organic solvent
> electrostatic interactions are much stronger than in water
> stabilise TS or guide substrate entry
types of catalysis: cat. through proximity and orientation
2/3 ig with eg of how
reactants req. proper orientation for reaction
> better orientation = better reaction rate
e.g. with optimal orbital overlap
types of catalysis: preferential TS binding/stabilisation
inc conc of TS = inc reaction rate
> enzymes usually favour alt pathway
types of catalysis: preferential TS binding/stabilisation
TS analogues?
TS analogues are compounds that resemble TS key properties but are stable
> these are generally potent enzyme inhibitors !
Proteases:
what it is + about the peptide bond
cat. peptide bond hydrolysis
peptide bond = metastable so hydrolysis is exergenic and releases energy (E leaves)
without cat. = very high Ea and kinetically trapped
the 4 groups of proteases (similarities and differences) and small explanation
based on active site architecture, all undergo nuc. attack on Carbonyl but with a different Nucleophile
Serineproteases: nu = serine hydroxyl O
Cysteineproteases: nu = Cys thiol (s) activated by His
Aspartate: nu = H2O activated by Asp.
Metalloproteases: nu = H2O AND substrate both of which are activated by divalent metal ion. e.g Zn 2+
Endoproteases: what does
opposite?
cleave internal peptide bonds (inside chain) by recognising specific cleavage site sequences !!
Exoprotease = opposite = cleaves terminal peptide bonds
nomenculture of protease substrate specificity
numbering relative to cleaved bond
P1,P2, P3, P4 to N-terminus (from C=O then)
P1’, P2’, P3’, P4’ to C-terminus (from N-H then)
Endoproteases recognition seq.
2
specificity achieved through specific binding pockets, and recognition sites distinguished by residues at bottom
Serine proteases
active site build? superfamilies? how many and explain how they come
share common active site build: Asp-His-Ser catalytic triad !
there are 16 superfamilies of serine proteases based on… (protein folds)
> active site arrangement which can differ within the sequences of different serine protease families (always asp-his-ser but located at diff parts)
same active site can therefore results in various different protein folds!
> complex structures don’t have to be related despite having same active site
> diff 3D topology, diff active site residue positions in AA sequence
Serine proteases mechanism
and explain acid-base cat.
all share common mechanism
2 parts:
1: peptide bond cleavage and acyl-enzyme intermediate formed
> Ser Hydroxyl (base cat.) attacks carbonyl which results in tetrahedral TS that eventually is acid hydrolysed to
> Acyl-enzyme intermediate: N-terminal attached to serine
> release of C-terminal (this is the NH- side)
2: Hydrolysation/Deacylation of the acyl-enzyme IM
> H2O req. (base. cat) which attacks carbonyl forming tet. TS again and acid cat required to:
> release of N-terminal
> restoration of ser-enzyme
acid = donating H+
base = accepting H+
so first the ser-oh is base cat. bc it’s H+ is removed to make it O-
but later the NH- requires H+ so it is acid catalysed
same thing happens with H2O and CO- later
small reminder about c-term/n-term
c-terminal is on the NH side of the peptide bond as if u go down the seq. it will end with COOH
n-terminal is on the C=O side of the peptide bond as if u go down the seq. it will start with NH3
NH2——CONH——-COOH
bit counterintuitive but it works if u think abt the bigger picture !
common features of serine protease active sites
4 + small details
cat. triad: Asp-His-Ser
oxyanion hole for stab/tight binding of tetrahedral TS
unspecific backbone/main chain to help mediate substrate binding/recognition
specificity pocket near cleave site
more about the oxyanion hole
tetrahedral TS stabilisation: -ive charge stabilised by Hydrogen binding to two backbone NH (+’ve)
Trypsin: what is it/where
2
digestive serine protease
produced in pancreas as proenzyme: tripsinogen
> activated by proteolytic cleavage
Bovine pancreatic trypsin inhibitor (BPTI)
general abt PTI
what is BPTI
how does it work
pancreatic trypsin inhibitor binds to misactivated trypsin as a fallback mechanism: prevent unwanted dmg
compact small protein that binds very tightly to trypsin
by having substrate like interactions, provides scissile bond that almost forms tetrahedral state
> but this state is sterically restricted
> water can not enter
> reaction does not continue ! (cant hydrolyse)
Trypsin substrate recognition
specificity pocket + peptide recognition by main chain forming H-bonds
requires correct alignment and position to activate the first step of protease … (Ser OH deprotonation)
