Enzymes Flashcards
What are the effects of pH on enzyme activity?
Activity is dependent upon pH of medium
Enzymes are only active over a narrow pH range: Essential groups are either protonated or unprotonated in order to react with substrate.
Maintenance of correct conformation for catalysis dependent on?
on ionisation of amino acids side chains
Catalytic amino acids residues often have?
ionisable functional groups: charged (unprotonated) acids, protonated bases (Thiol, imidazole, etc)
Effect of pH on kinetic constants:
Possible to identify active site amino acids by?
Examining effect of pH and Km and Vmax
Effect of pH on kinetic constants: Ionsiable groups in the active site will produce?
Titration curves for Km and Vmax
Amino acid side chains essential for catalysis alter Vmax
Amino acid side chains essential for binding alter Km
Effect of pH on kinetic constants: Amino acid side chains essential for?
catalysis alter Vmax
binding alter Km
Effect of pH on kinetic constants: Identification is only?
Tentative
Effect of pH on kinetic constants:Tentative meaning?
pH effects on the formation of the enzyme
pKa of side chains in proteins is often different to that in free solution
Example: pKa of Arg and Glu side chains can be increased in proximity to hydrophobic residues
Glu35 in lysozyme is protonated at pH 5
Enzymes catalysed reactions are highly specific meaning?
A given enzyme will always catalyse the same reaction
Specificity is normally absolute
Some enzymes work on classes of compounds
Substrates can be one of many hexose sugars
Specificity and mode of catalysis are determined by active site
What is the active site of an enzyme?
Region of the protein essential for catalysis
Composition determined by whole protein
Comprises a small fraction of total protein: Typically, a fold/cleft between domains or sub-units.
What are the molecular features of the active site?
The surface is lined with amino acid residues responsible for substrate binding and catalysis
Comprised mainly of hydrophobic residues:
Several polar and charged species also present
These are frequently catalytic residues
What constitutes the catalytic centre of the enzyme?
Polar residues (and coenzymes) which undergo chemical changes during catalysis constitute the catalytic centre of the enzyme
What are the type of reactions the residues participate in are nucleophilic or electrophilic substitutions?
Nucleophile: Molecular species is electron rich
Negatively charged, or has an unshared electron pairs
Electrophile: Electron poor
Positively charged or has a slight positive dipole
What are the ionic intermediates?
Acyl group transfer
Direct displacement reactions
Explain lock and key hypothesis
Enzymes are rigid locks to which the substrate is a precisely fitting key
3-D shape of the active site is complementary to 3-D shape of the substrates
Explain Induced fit hypothesis
Enzymes are flexible molecules
Binding of substrate causes conformational change in enzyme resulting in catalysis
Explain increasing local concentration of substrates
Enzymes act as entropy traps by collecting highly mobile reagents from dilute solution
Decreases entropy
Increases probability that reaction will occur
Explain correctly orientating substrates for reaction to occur
Reducing degrees of freedom of interaction
Reduces entropy
Increases likelihood of reaction occurring
Explain Exertion of strain on covalent bonds
Lowering activation energy for bond cleavage
Physical distortion (bond strain)
Changes to charge distribution within substrate
Define transition state hypothesis
Enzymes are flexible molecules, the shape of which is precisely complementary to the 3-D structure of substrate(s) in the activated “transition state” geometry
Rate reaction is dependent upon?
Reactant interactions: Both concentration and free energy of reactants are important in this
Transition state hypothesis: When do products form?
Products form when reactant collision reaches a configuration resembling the transition state of a reaction
How do enzymes catalyse reactions?
By stabilizing the transition state:
Catalysts provide reaction pathways which have lower activation energies (Delta G) compared to uncatalysed reactions (Delta G).
What are intermediates?
Reactions often proceeds via formation of intermediates
They are products formed between the final product
Define Acid-Base catalysis
Acceleration of reaction rate achieved by catalytic proton transfer
Commonly found mechanism of enzymatic catalysis
Amino acids which are can donate or accept protons at near neutral pH are important in this type of mechanism.
Proton acceptors are bases (B:) or ionised acids (A-)
Proton donors are the conjugate acid of a base (BH+) or protonated (uni-ionised) acids (AH).
What are the two ways Proton acceptors can assist in catalysis?
Direct cleavage of O-H, N-H or 9some) C-H bonds
Cleavage of other carbon-containing bonds (e.g C-N) by generating OH- equivalents from water in neutral solutions
How can proton donors assist in catalysis?
Proton donors (BH+ or AH) can also assist in bond cleavage
Covalent bonds may break more easily if one of the atoms is protonated by a general acid.
BH+ or AH can catalyse bond cleavage by donating a proton to an atom more labile
Define Covalent catalysis
A substrate, or a part of a substrate is bound covalently to an enzyme to form a reactive intermediate
Subsequent steps in the catalytic mechanism transfer reactive intermediate to a second substrate
