Active Site - Characteristics
Active Site = Substrate Binding site + Catalytic site
1. aa residues are not necessarily adjacent in primary sequence but are brought into position to form the active site by the protein's folding pattern.
2. Is a small portion of the enzyme.
3. Forms a cleft/pocket for substrate.
4. Has specificity for the substrate, depending on precise arrangement of atoms in the active site.
Binding of Substrate(s) - Characteristics
1. 3-D geometry of binding site, spatial considerations & orientation:
a. Protein folding pattern must form cleft/pocket to fit size & shape of substrate.
b. Steric hindrance limits access.
c. Chiral properties limit orientation.
2. Weak interactions between substrates & enzyme's aa side chains: hydrophobic, H-bonds, electrostatic, van der waals
3. Transient covalent bonds may form between the developing intermediate and enzyme aa side chains (covalent catalysis).
4. Order of binding & release of substrates and products
Cleaves after long, positively-charged side chains: Arg, Lys
Cleaves after aromatics: Phe, Tyr, Trp
Theories of E-S Binding
1. Lock & Key Model: specifically complements. BUT not ideal for catalysis - no room to move around
2. Simple-Induced Fit Model: Induces conformational changes in enzyme. Pauling - should fit substrate but should be complementary to TS to lower TS energy. (Ex: Oxygen in Heme)
Proximity & Orientation
Occurs for all enzymes
General Acid/Base Catalysis
Enzyme will donate/steal proton and will be regenerated by stealing/donating another proton.
Transient covalent bond (Cys, Ser, His, Asp, Glu, Lys, Arg) - makes a Schiff base via a Lysine side chain (NH2). Net reaction will be the same but rate of reaction is enhanced due to covalent E-S intermediate.
Ex: Covalent catalysis stabilizes oxonium ion.
Schiff base: (R1)(R2) - C = N - (R3)
Mediated by aa side chains that are charged at physiologic pH (Glu, Asp, His, Lys, Arg). Can also be mediated by metal ions.
Promotes catalysis by:
1. Stabilization of binding & orientation of +/- charged substrates
2. Stabilization of +/- charged reaction intermediates
3. Guiding/propulsion of polar/charged substrates into binding sites
4. Electrostatic interaction between two or more charged side chains of the enzyme can also promote catalysis in some cases
Metal Ion Catalysis
Two classes of metal-requiring enzymes: (all positively-charged)
1. Metalloenzymes: tightly bound metal ions (Fe2+/3+, Cu2+, Zn2+, Mn2+, Co3+)
2. Metal-activated enzymes: loosely bound (Na+, K+, Mg2+, Ca2+)
Promotes catalysis by:
1. Serving as redox agents (such as Fe-S clusters, heme iron, Cu ion)
2. Electrostatic effects:
-Stabilize binding & orientation of negatively-charged substrates
-Stabilize negatively-charged reaction intermediates
-Shield/neutralize negative charge density on substrates (such as Mg-ATP)
-Act as Lewis acids by 1) hyperpolarizing bonds to cause deprotonation at neutral pH, and 2) acting as an electron sink by withdrawing electrons from a carbonyl carbon to enhance its partial positive charge and make it a better site for nuc attack.
3. Guiding charged substrates into the active site
Preferential Binding of TS complex or reaction intermediate
1. Electrostatic stabilization of developing charge on intermediate (Ex: oxyanion hole to stabilize negative charges)
2. Relief of bond angle strain and enhancement of weak interactions between Enzyme & TS
Ex: Steric hindrance of C6 of NAM upon binding to subsite D (by lysozyme sidechains) forces C6 into the axial position, causing strain on the NAM ring which distorts it into the half-chair conformation. This induced strain makes binding at subsite D unfavorable, even though the net binding constant is quite favorable. Cleavage between D & E site leads to resonance-stabilized cationic TS that prefers the half-chair conformation (b/c the anomeric carbon is no longer tetrahedral - It now binds to subsite D without strain). This releases the strain and leads to tighter binding of the TS than of the substrate.
Catalytic Triad - Chymotrypsin, a Serine Protease
Asp (Coo-), His (ring structure w/ potential N+), Ser (pKa = 13, too high to be in unprotonated state?).
When peptide binds, it causes a conformational change that suppresses the H-bonding network. It increases pKa of His to 12, so that His can act as a general base. It also prevents Ser from developing an unstable positive charge after its proton is taken by His.
1. Stabilization of His as a good base (low H-bonding changes pKa to 12).
2. His deprotonates Ser to make Ser a good nuc.
3. Ser attacks carbonyl carbon on substrate.