BIOC 503 - General Enzymology I

catalysts

enzymes are ___, they increase the reaction rate without being used up.

RNA

Most enzymes are globular proteins, though some ___ (ribozymes and ribosomal) can also catalyze reactions.

advantages of biocatalysts

greater reaction specificity (avoid side products)

Also:

• milder reaction conditions (adapted to conditions in the cell)

• higher reaction rates (in biologically useful timeframe)

• capacity for regulation aka control of biological pathways

favorable

enzymes make the most desired metabolites the most ___.

active site

enzyme-substrate complex drives selectivity, AKA ___ drives selectivity

equilibrium, free energy

enzymes DO NOT affect ___ (Keq)

• if complex formation rate increases, then complex de-formation also increases so the ratio aka Keq stay the same

• It also means that enzyme CANNOT affect the ___ of the reaction

activation energy

enzymes work by decreasing the ____ barrier of the reaction

• correspond to the distance from the initial coordinates to the transition state peak

transition state

the peak of a reaction coordinate diagram corresponds to the transition state

enzyme-substrate, enzyme-product

For a catalyzed reaction, the first bump corresponds to the ___ complex, and the second bump correspond to the ___ complex.

proximity, orientation

enzymes work by organizing reactive groups into close ___, and proper ___.

• they basically position everything into the ideal position.

bimolecular

uncatalyzed ___ reactions are unfavorable because getting the free reactants together is where the entropy cost is

unimolecular

uncatalyzed ___ reactions are unfavorable because making the flexible reactant into something more rigid is where the entropy cost is

stereochemically  

when reactant are ___ restrained y enzyme where no rotation possible and sitting right next to each other with no possible movement lead to the highest rate in catalyzed reactions.

complimentary, better, stonger/additional

enzymes bind to transition states best because:

• active sites are ___ to transition state

• transition state binds ___ than substrate

• ____ interactions with transition state lower activation barrier

substrate

if the enzyme were most complimentary to the __ then it would happily bind, but NO reaction would occur

• That’s why enzyme partially bind to ___, and full binding does not occur until the transition state so reaction can proceed.

acid-base catalysis

Basically, give and take protons.

• Because water ___ functions are limited by pH significantly, which is why ___ is usually carried by amino acids like Glu, asp, Lys, Arg, his, Cys, ser, Tyr.

• charge development avoided by donation of protons from HA OR proton abstraction using B:

covalent catalysis

Basically, change reaction path by forming a transient covalent bond between enzyme and substrate

• REQUIRES NUCLEOPHILE on the enzyme (can be a reactive serine, thiolate, amine, or carboxylate).

metal ion catalysis

Basically, use redox cofactors and pKa shifters.

• involve metal ion bound to the enzyme

• metal ion interact with substrate to stabilize negative charges

• participate in oxidation reactions

Chymotrypsin

One of several proteases that cuts peptide bonds during digestion.

• Cut peptide bond next to aromatic amino acids on C-terminal side

• Lives outside the cell in oxidative environment, thus is stabilized by disulfide bonds.

• performs catalysis using a catalytic Triade (Ser195, His57, Asp102, Gly193)

catalytic Triade

3 main amino acids carrying catalytic functions of chymotrypsin

• Ser 195

• His 57

• Asp 102

(Also, Gy 193, but it mainly stabilizes intermediate and is not directly involved).

Asp102

Aspartate residue of catalytic Triade of chymotrypsin.

• in carboxylate (negatively charged form) and thus H bonds with one nitrogen atom in imidazole group of histidine residue His57

• position His57 properly for catalysis

His 57

histidine residue in chymotrypsin’s catalytic Triade with a pKa near 6.5.

• second nitrogen in imidazole group protonated by the hydroxy group of Ser195, creating a hydroxide (which is stabilized by Gly193 and the nitrogen in the backbone of Ser195)

• carries out acid/base catalysis

• second imidazole N deprotonated when C-N bond in substrate later breaks, and nitrogen grabs proton.

• second imidazole N re-protonated by water

• ultimately deprotonated and back to normal when serine O- grabs extra proton

Ser195

serine residue of the catalytic Triade of chymotrypsin.

• carries covalent catalysis

• once hydroxy group deprotonated by histidine, new hydroxide acts as nucleophile and covalently bond to carbonyl carbon in substrate, creating a negatively charged oxygen (which is stabilized by Gly193 and nitrogen in serine backbone) and a tetrahedral carbon.

• carbonyl reforms, breaking the C-N bond in intermediate/substrate and deprotonating His57, then H2O comes in to act as nucleophile and re-protonating His57, creating another new hydroxide binding to carbonyl carbon and creating negatively charged O.

• carbonyl reform, leading to Serine bond to carbon breaking and serine grabbing proton on his57 —> enzyme back to normal

Chymotrypsin mechanism

1- Asp102 H bond to His57 to position it properly

2- His57 protonated by hydroxy group of Ser195. Created O- binds to carbonyl carbon on substrate, creating a new O- intermediate

• stabilized by Gly 193 and N in Ser195 backbone

3- carbonyl reforms, breaking substrate’s C-N bond, and leaving nitrogen deprotonate His57. Dissociation of product 1.

4- water acts as nucleophile and protonate His57 before hydroxide binds to substrate tetrahedral carbon, creating another O- intermediate

• stabilized by Gly 193 and N in Ser195 backbone

5- Carbonyl reforms again, leading to the link between Ser195 and intermediate breaking. Ser195 deprotonate His57, and enzyme back to normal after dissociation of product 2.

peptidoglycan

polysaccharide found in many bacteria cell walls

lysozymes

antibacterial enzymes binding to peptidoglycan and cleaving the bacteria cell wall by breaking B1-4 linkages

• aka lysis of bacteria

• Asp52 acts as nucleophile in first Sn2 step, Glu35 acts as general acid protonating leaving group, and Glu 35 then act as base to deprotonate water in second Sn2 step