(9-12) Proteins & Enzymes

What are the four major types of macromolecules in cells?

Proteins nucleic acids carbohydrates and lipids

How many proteogenic amino acids are there?

20 standard amino acids

What configuration do all amino acids have except glycine?

L-configuration

Which amino acid is achiral?

Glycine

Which amino acid has R-configuration instead of S-configuration?

Cysteine

What is the charge state of aspartic acid and glutamic acid at physiological pH?

Conjugate bases or carboxylates with negative charge

What is the charge state of lysine and arginine at physiological pH?

Conjugate acids meaning protonated with positive charge

How many amino acids do proteins typically contain?

100 to 300 amino acids

What type of bond connects amino acids in proteins?

Peptide bonds or amide bonds formed by condensation reaction

In which direction are proteins synthesized?

From N-terminus to C-terminus

What are polymorphisms?

Subtle differences in protein sequence between individuals arising from DNA variations

How can polymorphisms affect drug therapy?

They change the rate at which drugs are processed and excreted affecting drug potency and dosing

What are the four levels of protein structure?

Primary secondary tertiary and quaternary structure

What does primary structure refer to?

The sequence of amino acids from N-terminus to C-terminus

How many amino acids per turn in an alpha-helix?

3.6 amino acids per turn

What type of bonding stabilizes alpha-helices?

Hydrogen bonds within strands between C=O and N-H groups

What are the two types of beta-sheet arrangements?

Parallel running in same direction and anti-parallel running in opposite directions

What is the primary driving force for tertiary structure formation?

Hiding hydrophobic regions from water driven by entropy

What forces stabilize tertiary structure?

Hydrophobic interactions hydrogen bonds van der Waals forces salt bridges and disulfide bridges

What is quaternary structure?

Two or more peptide subunits held together usually by non-covalent bonding

What are common examples of quaternary structure?

Dimers with 2 subunits trimers with 3 subunits and tetramers with 4 subunits

What are enzymes?

Proteins that catalyze conversion of substrates to products

Do enzymes change the equilibrium position of reactions?

No they do not change equilibrium but speed up rate

What are the two models of enzyme catalysis?

Lock and key model and induced fit model

Which model is particularly important for multi-substrate enzymes?

Induced fit model

How do most enzymes achieve catalysis thermodynamically?

By lowering activation energy of the reaction

What type of chemistry is particularly important in enzyme reactions?

Carbonyl chemistry

What is the role of active site hydrophobic residues?

They exclude bulk solvent increase charge density and alter pKa values of charged residues

What do kinases do?

Add phosphate groups to substrates

What do phosphatases do?

Remove phosphate groups from substrates

What are hydrolases?

Enzymes that split compounds using water

What are proteases?

Enzymes that hydrolyze protein peptide bonds

What mechanism do serine proteases use?

Acyl-substitution mechanism with formation of acyl-enzyme intermediate

Which amino acid acts as nucleophile in chymotrypsin?

Serine with its hydroxyl group activated by histidine

What is formed during Step 2 of chymotrypsin mechanism?

Acyl-enzyme intermediate after tetrahedral intermediate collapses

How is the acyl-enzyme intermediate resolved?

Water acts as nucleophile to hydrolyze and regenerate free enzyme

What is Km in Michaelis-Menten kinetics?

Substrate concentration at which reaction velocity equals 0.5 times Vmax

What is Vmax?

Maximum velocity when all enzyme active sites are occupied

What does kcat represent?

First-order rate constant for conversion of substrate to product equal to Vmax divided by enzyme concentration

What does kcat/Km measure?

Catalytic efficiency of enzyme allowing comparison between enzymes

What order kinetics do enzymes follow at low substrate concentration?

First-order where rate increases with substrate concentration

What order kinetics do enzymes follow at saturating substrate concentration?

Zero-order where rate is independent of substrate concentration

What does the Lineweaver-Burk plot graph?

1/v against 1/[S] to determine Vmax and Km

What is the Y-intercept in Lineweaver-Burk plot?

1/Vmax

What is the X-intercept in Lineweaver-Burk plot?

-1/Km

In competitive inhibition which parameter changes?

Km increases while Vmax remains unchanged

Why does substrate overcome competitive inhibition?

Inhibitor competes for active site so high substrate outcompetes inhibitor

In non-competitive inhibition which parameter changes?

Vmax decreases while Km remains unchanged

Where does non-competitive inhibitor bind?

To site other than active site reducing catalytic activity

What characterizes uncompetitive inhibition on Lineweaver-Burk plot?

Parallel lines because both Km and Vmax decrease by same factor

Where does uncompetitive inhibitor bind?

Only to enzyme-substrate complex not free enzyme

What is IC50?

Concentration of inhibitor required for 50% reduction in activity at fixed substrate concentration

Is IC50 the same as Ki?

No IC50 depends on assay conditions but can be used to calculate Ki

What percentage of drugs are enzyme inhibitors?

Approximately 50% of drugs in human medicine

What is rational drug design for enzyme inhibitors?

Designing molecules that mimic substrate or transition state based on enzyme mechanism knowledge

What are tight-binding inhibitors?

Inhibitors with very slow dissociation rates allowing infrequent dosing

What is an advantage of tight-binding inhibitors?

Long half-life of enzyme-inhibitor complex allowing less frequent dosing

What is a disadvantage of tight-binding inhibitors?

Slower onset of action

What type of inhibitors are penicillins?

Irreversible inhibitors that form acyl-enzyme intermediates with penicillin-binding proteins

Why do irreversible inhibitors inactivate enzymes permanently?

They act as pseudo-substrates that get stuck at intermediate stage and cannot complete catalytic cycle

What enzyme do aspirin and ibuprofen inhibit?

COX-1 and COX-2 enzymes involved in inflammation

What enzyme does captopril inhibit?

Angiotensin-converting enzyme or ACE for blood pressure control

What factors affect enzyme activity?

pH temperature substrate concentration amount of enzyme presence of inhibitors and denaturing agents

How does pH affect enzymes?

Changes ionization state of side chains and extreme pH causes protein unfolding

What happens at temperatures above enzyme optimum?

Thermal denaturation occurs typically 2-3 degrees Celsius above optimum

What are denaturing reagents used for?

To unfold proteins and quench enzyme reactions examples include urea detergents and guanidinium hydrochlorid