5-6: ENZYME MECHANISMS Flashcards
bronsted acid and bases
BA: species that donates H+; HA is acid; A- is conj. base
BB: species that accepts H+; B is base; BH+ is conj. acid
why do acids and bases catalyse reactions?
- if the acid/base can interact w/ the molecule during the TS they can help neutralise charges that develop during bond breakage because they can deliver or abstract protons
- this results in reducing gibbs free energy of activation
general acid catalysis
process whose rate of reaction is dependent on the concentration of all acids present in the reaction, not just the concentration of protons
general base catalysis
process whose rate is dependent on concentration of all bases present in reaction, not just concentration of hydroxide
specific acid catalysis
reaction just dependent on concentration of protons not any added acid
specific base
reaction dependent on concentration of hydroxide ions, not any added base
general acid-base catalysis
e.g. keto-enol tautomerisation
UNCATALYSED: in the transition state, the molecule separates charge through breakage of C-H bond; this is disfavored because opposite charges are building up; this makes overall uncatalysed reaction slow
GENERAL ACID CAT: add HA which reduces energy of TS by delivering a proton to the O as its accumulating charge; this enhances rate of reaction
GENERAL BASE CAT: add B which starts to pull of proton which reduces energy level of TS as it reduces degree of charge separation
ph dependancy of acid-base catalysed reactions
- enzyme shows activity dependent on the concentration of acid in the enzyme
- can determine pka value: pH at which there is 50% activity
- reversible inhibition: can increase pH to deprotonate and switch off the enzyme and then reacidify the assay to protonate the enzyme and activity comes back
- pH optimum: need acid and base in right protonation state for enzyme to be active (B and HA)
- pH profile of enzyme activity is bell shaped
effect of environment on pka values
- enzymes control the pKa value of active site by manipulating the microenvironment
- hydrophobic region; uncharged species favoured; pKa I
- negative charge nearby; positive charge stabilised; pKa I
- positive charge nearby; negative charge stabilised; pKa D because Ka I due to formation of conj. base will be enhanced because of stabilising effect of charge-charge interactions (e.g. glutamic acid and lysine in active site of enzyme)
covalent catalysis definition
- enzymes provide lower energy pathway by forming covalently bound enzyme intermediates that form from reaction of substrate with an amino-acid side chain or a co-enzyme
- Asp, glu, his, cys, tyr, lys, ser, thr are potential nucleophiles; only reactive when deprotonated as they need LP of e
- often E activate their Nu by providing a base close by that will pull off proton and free LP of e
- in this mechanism, have E and in active site a Nu (ie. a side chain) to perform Nu attack on substrate to form a covalent bond and help catalysis
where is covalent catalysis used
- in group transfer reactions
- to make the substrate more reactive
what are group transfer reactions
- bisubstrate reactions where substrate A and substrate B come together in the active site and react
- during course of reaction, groups are transferred
- could be reactive group ie. amino group NH2 or acyl group R-C=O
- sometimes unreactive group ie. phosphoryl PO3,2- (ATP is a carrier of this)
group transfer reaction via ternary complex
- binding substrates A and B into the active site to form ternary complex (E+A+B)
- group is transferred there; product forms and is then released
- could occur in ordered process or random binding and release
group transfer reaction via ping-pong mechanism
- relies on covalent catalysis
- ordered process
1. binding of A in E with Nu and forms EA complex - nucleophilic reaction; group is transferred to side chain or co-enzyme
- forms product P and modified enzyme F which now has group
- product released; only have F
2. second substrate comes into active site - forms FB complex
- group is transferred to B to form Q
- have EQ complex and then Q is released to reform E and can start catalysis again
making substrate more reactive in covalent catalysis
- have E with Nu
- nucleophilic attack on S
- forms covalent bond and activated form of S, S*
- can now form P which is still covalently attached to E through amino acid side chain
- bond breaks between Nu and P to release P and regenerate E
activating carbonyl compound
- improve ketone reactivity by converting to schiff base (are analogous)
- ketone + primary amine
- addition elimination reaction where water is removed
- more reactive because its a more polarised system
- LP of e still available on N and can be protonated by general acid to generate protonated imine which is even more reactive
- can draw resonance structure, +ve charge on C and neutral N or vice-versa; pi cloud is shifted towards nitrogen to make v. electrophilic C
acetoacetate decarboxylase enzyme
- produces acetone and carbon dioxide
- catalyses bond breakage reaction
UNCAT:
- usually formation of carbanion is disfavored because of high energy but now have carbonyl group which acts as e sink
- electronegativity of O in carbonyl group affords some stability to the system because it likes accumulating some negative charge; this aids in breakage of C-C bond (move e into O)
- end up with enolate anion which helps stabilise the formation of the carbanion through resonance stabilisiation
- problem: still have -ve charge in the system
CAT:
- lys (only aa with primary amine functionality) reacts w/carbonyl group to generate protonated schiff base (enamine) which is neutral and thus more stable so TS energy is lower as less charge is accumulating
- better e sink
how to detect formation of schiff base in an enzyme
- chemical trapping of enzyme intermediates
- treat with NaBH4 (sodium borohydride); a source of H-, a v. strong nucleophile and will react with protonated schiff base
- N receives e and becomes neutral
- unreactive so cannot go through catalytic cycle
- stuck as modified form of lys side chain
- then digest E and identify which lys residue has been modified and thus gain details of residue involved in covalent catalysis
electrostatic catalysis definition
-prescence of charges and oriented dipoles within the active site can stabilise the transition state through solvation/electrostatic interaction (+ve with -ve)
what affects electrostatic interaction
- the greater the charge, the greater the energy of interaction
- the further apart the charges, the lower the energy of interaction
- polar molecules w/permanent dipole (e.g. water- high value of er), reduces the electrostatic interaction
- coulombic interactions are more effective in non-polar environments
- enzymes can fine-tune microenvironment to improve charge stabilisation because of the differences in dielectric constant of residues
types of metal ion catalysis
- two classes of metal ion requriing enzymes distinguishable by strength of binding:
1. metalloenzymes contain tightly bound ions (eg. Fe2+, Mn2+) through dative/coordinate bonds; always present
2. metal-activated enzymes loosely bind ions from the solution (e.g. Na+, K+); come in with S and help in catalysis then go out with S
what are metal ions involved in
1) electrostatic stabilisation and charge screening; highly effective due to high charge that is pH independent e.g. Mg2+-ATP
2) redox reactions: lots of transition metals can be ox and red so are involved in transferring e in the cell (e transport pathways to oxidise NADH and convert proton motive force to generate ATP)
3) can act as lewis acids so can accept LP of e and can bind e donor in a dative/coordinate covalent bond; this determines the orientation of S so it reacts in most favourable geometry
4) promote nucleophilic catalysis by activating bound water molecules (e.g. Zn2+ ion in carbonic anhydrase); helps activate relatively inert molecules due to its charge
Mg2+-ATP complex in metal ion catalysis
- ATP usually at physiological pH carries 3-4 negative charges which can be big hindrance
- ATP always used as a substrate in Mg-complex
- Mg2+ associates w/ATP and goes into active site; it offsets some of the -ve charge of ATP to allow nucleophilic attack on it
redox roles in metal ion catalysis
- e.g Fe2S2 iron clusters
- dative bond to cysteine residues (2 e from cys are donated to Fe)
metal ion catalysis reedox eg. Complex I (NADH: ubiquinone oxidoreductase)
-NADH is being oxidised; ubiquinone is being reduced
-enzyme found in mitochondrial membrane
-has large hydrophilic arm that sticks out
-UQ is v. hydrophobic; exists in membrane; comes into active site and is reduced to UQH2 (ubiquinol)
-UQ + 2e + 2H+ = UQH2
-NADH is e carrier/donor which goes into site in hydrophilic arm
-about 8 FeS clusters so e can hop through
-during this process, protons are pumped across the membrane
-
metal ion catalysis redox e.g. Photosystem II
- oxidises water to produce O2 + 4H+ + 4e with help of light
- 4e used to fix CO2 for growth
- the cluster is made of Mn4CaO5
- multiple metal ion centres to accept e
metal ion catalysis activating water e.g. carbonic anhydrase
- OH- is v.good nucleophile but too little of it
- to increase OH-, bind water to metal ion
- CO2 + H2O = HCO3- + H+ involves Nu attack on CO2
- in carbonic anhydrase enzyme, there is Zn2+ permanently bound into active site because it is ligated by 3 residues; has one empty position which is occupied by a water molecule and forms coordinate bond
- Zn2+ and OH- there is a charge-charge interaction so there is now stabilisation and pKa could be as low as 7 so at physiological pH now have high accumulation of OH-
- water pKa of 15-16 so mainly protonated form, v.little OH-
catalysis through orientation and proximity effects definition
- increase rate of reaction by taking 2 reactants out of solution and placing them next to each other in the active site of an enzyme thus raising the local concentration of each reactant
- A and B need to be in right orientation so they can form bonds w/each other
entropy changes in bimolecular reactions
- uncatalysed: ^S large -ve value = highly unfavourable because are forcing order
- catalysed: ^S smaller -ve or +ve = more disordered = more favourable due to binding in active site (split up reaction in 2 steps)
preferential binding of transition state enzyme catalysis
- E complementary to TS not S to maximise interaction and reduce gibbs free energy of interaction
- much better than E-S complementarity
how do enzymes catalyse reactions
- uncatalysed reactions are slow because many molecules come together in TS (loss of entropy) and unstable +ve and -ve charges develop in the TS
- enzymes function to provide strategically placed acids, bases, metal ions and dipoles to stabilise charges
- provide alternative lower energy reaction pathways through covalent catalysis
- reduce effect of entropy by incorporating catalytic groups as part of the enzyme
