exam 4: enzyme mechanisms and regulation

what are the 4 mechanisms of catalysis

1. covalent catalysis

2. acid-base catalysis

3. low-barrier hydrogen bonds

4. metal ion catalysis

what are the biochemical nucleophiles

• serine (-OH)

• cysteine (-SH)

• lysine (-NH2)

• histidine (imidazole group)

• water (H2O)

what is a nucleophile

an electron-rich species that donates a pair of elections to an electrophile to form a chemical bond

chymotrypsin, a protease enzyme uses __

both covalent and acid/base catalysis

what is covalent catalysis

the enzyme forms a termporary covalent bond with the substrate, creating a more reactive intermediate that facilitates the reaction

what does chymotrypsin cleave

the carboxyl side of large hydrophobic or aromatic amino acids such as phe, met, tyr, and trp

what are the mechanistic steps of chymotrypsin

1. Substrate binds to enzyme

2. nucleophilic residues in active site attack an electrophilic center on S, creating a covalent bond

3. tetrahedeal intermediate is formed, the enzyme stabilizes the high energy transition state

4. product formed through rearrangement or bond cleavage

5. water attacks the intermediate to break the EP complex

6. product is released

what is the overall process of acid-base catalysis

a proton is transferred to catalyze reactions

what are the mechanistic steps of acid-base catalysis

nucleophilic attack of base, proton transfer, product forms

what is specific acid-base catalysis involve

H+ or OH- that diffuses into the catalytic center (strong acid or strong base)

what does the rate of a specific acid-base reaction depend on 

the pH of the solution, not concentration of the buffer

what does general acid-base catalysis involve

acids and bases other than H+ and OH-

what does the rate of a general acid-base reaction depend on 

depends on both the pH and the concentration of the buffer

what are examples of enzymes that employ general acid-base catalysis

serine and aspartic acid

what is a low-barrier bydrogen bond (LBHB)

special type of H bond where the proton is more equally shared between the donor and acceptor atoms, rather than being strongly associated with one

exceptionally strong and play a critical role in enzyme catalysis by stabilizing transition states and lowering activation energy

strength: 10-30 kj/mol

what are the mechanistic steps of LBHBs

1. S binds to E via H bonding: Asp, Glu, His, Tyr are common residues for H bonding

2. hydrogen bond strengthening, the H bond is shortened. transition state is stabilized

3. transition state collapses through bond cleavage or formation

4. LBHB reverts to normal H bond

what is metal ion catalysis

metal ions in an enzymes cofactors interact with the substrate, stabilizing the transition state and orienting the substrate for reaction

what are the mechanistic steps of metal ion catalysis

water activates

substrate binds

leaving group leaves

what are the 3 types of enzyme regulation

1. the presence of allosteric regulators or inhibitors

2. genetic regulation

3. compartmentation

what enzyme converts ATP into cAMP

Adenylyl cyclase

what is cAMP

the intracellular agent of extracellular hormones: a second messenger used in intracellular signaling (PKA activation)

how is cAMP formed from ATP

the 3’ hydroxyl on ribose attacks the alpha-phosphate of ATP, forming a 3’-5’ cyclic phosphate ring

why does breaking down pyrophosphate matter

hydrolysis of PPi is highly exergonic which drives the formation of cAMP forward and makes the reaction effectively irreversible 

what does the binding of Ga(GTP) to adenylyl cyclase do

activate cAMP production

what is cAMP-dependent protein kinase composed of

catalytic and regulatory subunits

what do the two regulatory subunits bind in cAMP-dependent protein kinase

two equivalents of cAMP each

cAMP binding releases the R subunits from the catalytic subunits

which amino acid side chain is involved with adenylation

tyrosine

which amino acid side chain is involved with uridylylation

tyrosine

which amino acid side chain is involved with ADP-ribosylation

arginine

which amino acid side chain is involved with redox

cysteine

which amino acid side chain is involved with acetylation

lysine

what is transcriptional control

gene expression can be upregulated or downregulated in response to cellular needs.

ex: lac operon in proks

what does the presence of lactose upregulate

the expression of lacZ, lacY, and lacA genes

why are enzymes, substrates, and regulatory molecules located in separate compartments/areas

so that opposing pathways are physicallyseparated, or located close together, to incraese pathway efficiency