Enzyme Catalysis PDF

Local environment can critically influence sidechain pKa - Typical Glutamate sidechain has a pKa of 4.1 - Lysozyme is active across a range of pH from \~4 to 9 - Average pH of human tears is \~7.4 - When: - pH \> pKa = deprotonated (molecule of the pKa in question) - pH \<...

Local environment can critically influence sidechain pKa - Typical Glutamate sidechain has a pKa of 4.1 - Lysozyme is active across a range of pH from \~4 to 9 - Average pH of human tears is \~7.4 - When: - pH \> pKa = deprotonated (molecule of the pKa in question) - pH \< pKa = protonated - Therefore, at the optimal pH for lysozyme (5), Glu sidechains would be negatively charged  All enzyme catalysed reaction occur in active sites on the enzymes Enzymes bind and organize substrates to react with each other in enzyme-substrate complexes Result - a maximum reaction velocity is reached when all the enzyme active sites are filled with substrate.   Enzymes enhance reaction rates through proximity and orientation effects- Most of the observed 10^8^ to 10^12^ rate enhancement comes from binding effects An enzyme effectively turns an otherwise **inter**molecular reaction into a faster **intra**molecular one. This is faster because all substrates are on the same surface and so it: - Bring substrates close together - Hold them in the optimal orientation so they react with each other   Rate enhancement as a result of controlled binding can be seen in model systems - Example: The reaction occurs when the negatively charged oxygen is positioned correctly near the target carbon 1. This is with no enzymes - just when reacting groups randomly bump into each other 2. An enzyme links the two carbons using a CH2 group, bringing reacting groups closer together. Still has rotation around the bond. Enhances reaction rate. 3. The two carbons are directly linked now. Less rotation (degree of freedom is reduced). Reacting groups are closer. Enhance rate of reaction. 4. The bonds are fixed. There is no rotation. Enhances reaction rate further. Summary: - As the reacting species are progressively held in more optimal alignment for the reaction to occur, the rate of reaction gets faster - This is what enzymes do by binding their substrates - All enzyme reactions can be considered as intramolecular   Intermediates and transition states lie on the path from substrate to product- - In going from substrate to product, all reactions pass through transition states and sometimes through intermediates - Difference between the two states is the time they last for (transition is very short) - transition states exist on very short femtosecond timescales (10^-13^ to 10^-14^) - Reaction intermediates are more stable than transition states and can be isolated   Transition state affinity- Enzymes prefer to bind to transition states and therefore facilitate their formation. Change the transition state barrier which reduces the activation barrier. A diagram of energy and energy Description automatically generated Many of the best enzyme inhibitors are transition state analogues (what we think transition states would look like) This idea has been exploited in the field of catalytic antibodies, and in inhibitor design. - Catalytic antibody - synthesise transition state analogue of the reaction you want to catalyse, raise antibodies to bind TS analogue -- antibody catalyses reaction (in an ideal world) - Inhibition - If an enzyme preferentially binds its transition state, then TS analogue should also preferentially bind over substrate and act as competitive inhibitor. Example - Proline Racemase: TS analogues bind to enzyme with 160-fold greater affinity than does proline substrate. In contrast tetrahydrofuran-2-carboxylate, which more closely resembles the tetrahedral structure of proline, is not nearly as good as an inhibitor as TS analogues.   Enzymes don't bind too tightly to their substrates- - Plot of the energetics of a reaction system and how that changes as the species change from substrate (S) to product (P) in the absence/presence of an enzyme. - Highlights the problem of binding a substrate too tightly in an enzyme substrate complex (ES) and how that can **raise** the activation energy barrier *S^‡^* (transition state) is favoured binding form of the molecule (i.e. reducing the activation energy, ΔG^‡^). If the substrate is very strongly bound following the **hypothetical dotted pathway**, then the activation for paths (1) or (2) would be comparable - we can see that the jump to get from the intermediate to the transition state is the same in each diagram so the enzyme does not benefit the system.   - Disadvantageous for enzymes to bind their substrates too tightly (evident when comparing substrate binding constants). - Typical values for binding constants catalytic enzymes are in milli- to micromolar range, whereas for binding proteins and antibodies, whose function is to bind substrate molecules tightly, are in the nano- to picomolar range. - Bigger values result in poorer binding   Enzymes are unchanged by the reactions they catalyse- - Enzymes are unchanged at the end of the reaction cycle - even when covalent intermediates are formed (these covalent bonds are then broken, usually by water) High specificity- - Active sites are optimised to make contacts with a substrate or range of substrates to enable catalysis - Enzymes exhibit substrate specificity based on size, shape and functional groups Lock and key\^\^ not representative of all enzymatic interactions   Enzymes can alter conformations to match more ideally to structures of substrates or transition states- Enzyme structure is flexible and enables optimal binding- - Substrate is bound non-optimally and is stressed (not using all possible interactions) when bound - Enzyme is strained (distorted) when substrate is bound, but this strain energy is relieved when transition state is reached, removal of this results in stabilisation of the transition state believed to be the major factor in reaction rate acceleration. - Induced fit model   Catalytic Mechanisms - Catalysis is a process that increases the rate at which a reaction approaches equilibrium. - Most of the rate enhancement in enzyme catalysed reactions comes from substrate binding and organization. However, it was found there was still some energy unaccounted for - these common steps which recur in enzyme catalysed reactions account for this: 1. General acid-base catalysis 2. Covalent catalysis 3. Use of metal ions in catalysis   1\. General acid-base catalysis - Involves donation of a proton by a group on the enzyme acting as an acid or - Abstraction of a proton by a group on the enzyme acting as a base *See written notes* for simple/general diagram Both processes lower the free energy pathway to the transition state in a reaction Acid and base catalysis may be combined in a single reaction cycle Example - Mechanism of action of lysozyme   - Glu35 (in an unusual protonated form at biological pH) donates a proton to the substrate and promotes cleavage of the glycosidic bond between R1 and R2 and thus **acts as an acid**. - Group HO-R1 can then leave the active site. - Glu35 then takes a proton from an attacking water and thus **acts as a base**. - The resultant HO- from the water can then attack R2 and complete the reaction cycle. - The net effect is that a water is added to break the R1-O-R2 bond.