Enzyme Catalysis and Regulation

• Enzyme catalysis stabilizes the reaction's transition state.
• Some enzymes require non-protein components (cofactors or prosthetic groups) for function.
• Enzymes are highly regulated by covalent modification and binding of non-substrate ligands (allosteric regulation).

Enzymes

• Enzymes are catalytic proteins that speed up cellular reactions.
• Enzymes do not alter the free energy change (ΔG) of a reaction.
• Enzymes cannot make a thermodynamically unfavorable reaction occur spontaneously.
• Enzymes cannot alter reactant or product concentrations at equilibrium.
• Enzymes do not extract more useful energy per mole of reactants, they only accelerate the process.
• Enzymes determine nearly all chemical transformations involved in making or breaking covalent bonds in cells.
• As catalysts, enzymes are required in small amounts.
• Enzymes must remain unchanged after a reaction to cycle back and bind more substrate.
• Enzymes catalyze forward and reverse reactions equally.
• Enzymes can increase reaction rates by 10^8 to 10^12-fold.
• Enzyme function begins with substrate binding at a stereo-specific active site, forming an enzyme-substrate complex.
• The active site has a 3-D shape that is particular to the substrate.
• After substrate binding, the substrate is chemically converted to product, forming an enzyme-product complex.
• The product is released, and the enzyme can bind another substrate molecule.
• Enzymes kinetics describe the rates of enzymatic reactions.

Vmax and Km

• Vmax is the maximum rate when the enzyme is saturated with substrate.
• Km (Michaelis constant) is approximately equal to the dissociation constant for the enzyme-substrate complex.
• A low Km indicates a high affinity of the enzyme for its substrate.
• The Km value of an enzyme usually lies within its substrate's natural concentration range.
• If the Km of an enzyme decreases, the binding interaction between the enzyme and substrate becomes stronger.
• A reduced Km also indicates that less substrate is required to half-saturate the enzyme.

Enzyme Binding

• Substrate molecules pass through intermediate states.
• The 3D geometry of reactants needs adjustment for optimal interaction.
• Electrons among reacting atoms become redistributed.

Transition States

• The transition state is an intermediate form between reactants and products, with a specific structure.
• Enzymes stabilize transition states, thus lowering the activation energy.
• As substrates react, they go through a state with a higher energy level.
• The energy needed to reach this state is the activation energy.
• Enzymes achieve transition state stabilization through various mechanisms.
• Enzymes bind two substrate molecules and orient them to encourage a reaction.
• Enzymes rearrange electrons in the substrate after binding.
• Rearranging electrons in the substrate creates partial negative and positive charges that favor a reaction.
• Enzymes strain the bound substrate molecule, forcing it toward a transition state.

Prosthetic Groups and Cofactors

• Prosthetic groups are non-protein molecules that aid in protein function.
• Prosthetic groups can be covalently or non-covalently bound by a protein.
• Hemoglobin uses heme as a prosthetic group for oxygen transport.
• Enzyme prosthetic groups are referred to as cofactors.
• Magnesium (Mg2+) is a critical cofactor for enzymes that join or cleave nucleic acids.
• Organic cofactors are often called coenzymes, such as coenzyme A (CoA).
• Vitamins frequently act as coenzymes or their precursors.

Molecular Tunnels

• Some enzymes perform multiple sub-reactions at distinct active sites.
• The enzyme's structure may act as tunnels to direct intermediate products from one active site to the next.
• The intermediates never leave the enzyme.
• Molecular Tunnels prevent diffusion of intermediates and decomposition of unstable molecules, speeding up reaction rates.
• Carbamoyl phosphate synthetase has 3 active sites connected by molecular tunnels.

Multi-Enzyme Complexes

• Most metabolic pathways require reactions in a specific, highly regulated order.
• Enzymes in a pathway can be organized into higher-order, multi-enzyme complexes.
• The product of the first enzyme is passed to the second enzyme as its substrate, and so on.
• Multi-enzyme complexes prevent product diffusion and allow for coordinated regulation of the pathway.

Enzyme Regulation

• Enzymes must respond rapidly to changing cellular conditions.
• Many enzymes are not constitutively active and are turned on/off when needed.
• Regulation of key enzymes involves multiple inputs, akin to molecular integrators.
• Feedback control regulates upstream enzymes: a downstream product regulates an upstream enzyme in a given pathway.
• When product concentrations reach sufficient levels, further production is halted via negative regulation.

Enzyme Inhibitors

• Enzyme inhibitors can be reversible or irreversible.

Irreversible Inhibitors

• Irreversible inhibitors covalently bind to an amino acid residue in the active site.
• Irreversible inhibitors usually lower Vmax by effectively inactivating and "removing" active enzyme molecules.
• Irreversible inhibitors are rare in nature but common in industry.
• Aspirin inhibits the enzymes COX-1 and COX-2 by acetylating a serine in the active site.

Reversible Inhibitors

• Reversible inhibitors are common in nature, and can be either competitive or non-competitive.
• Competitive inhibitors reversibly bind to the active site and compete with the substrate.
• Competitive inhibition can be displaced by high substrate concentrations.
• Competitive inhibitors increase Km, lowering the apparent "affinity," but they do not reduce Vmax.
• Non-competitive inhibitors reversibly bind away from the active site.
• Binding away from the active site causes a change in enzyme structure that lowers catalytic efficiency.
• Non-competitive inhibitors lower Vmax.
• Non-competitive inhibitors do not increase Km, and do not affect substrate binding.

Regulation of Enzyme Function by Covalent Modification

• Phosphorylation and dephosphorylation are common covalent modifications added to proteins to regulate activity.
• Phosphorylation allows for rapid, reversible regulation coded by a short (4-6) linear amino acid sequence.

Regulation of Enzyme Function: Allosteric

• Allosteric regulators are regulatory molecules with a unique shape, distinct from the enzyme's natural substrate.
• Allosteric regulators bind at a site away from the catalytic site.
• Binding of a molecule at one region of an enzyme influences the binding of another molecule to a different region via a conformational change in protein structure.
• The allosteric site is part of the same protein chain as the active site or in a "regulatory subunit".
• A small, allosteric molecule binds to a regulatory site.
• Binding results in a change in the conformation of the catalytic site.
• Conformational changes in the catalytic site can either activate (promote substrate binding/catalysis) or inhibit (prevent substrate binding/catalysis).
• Allosteric inhibitors possess qualities of competitive (affect substrate binding) and non-competitive (affect catalysis) inhibition.