Quiz 5 (L17-L18)

Construct a Go vs Progress of Reaction diagram for a given reaction mechanism (i.e., number of substrates and products, number and order of intermediates) and information about rate-limiting step, relative Gibbs free energy of states, relative rates of steps, etc.

• Greater concentration (favorable/unfavorable)

• Faster Formation (Favorable/unfavorable)

• Dissociation Constant > or < 1 (Favorable/Unfavorable)

• Stable (Favorable/Unfavorable)

The greater the x concentration the more favorable that x is (so lower G^o)

The faster the formation = The more favorable(so lower G^o)

Dissociation Constant < 1 Binding more favored(I,e ES)

Dissociation Constant > 1 Dissociation more favored(I.e E+S)

More Stable S or P is the lower G on graph

Overall TS is the highest G on graph

For chymotrypsin reaction pathway: why ES is higher energy than E+S

Enzymes drive the reaction, so it directly acts on the transition state and binding tightly.

• The substrate 6 peptide bond planar shape doesn’t fit well in the active site so it’s a higher energy ES(Tetrahedral intermediate is more favorable)

For chymotrypsin reaction pathway: what happens at step 1, given the blue boxes that precede and follow the step

1) Chymotrypsin Free Enzyme: No catalytic reaction occurs. Peptide planer/bond(substrate) comes in, but it doesn’t fit well to the active site.

Hydrophobic packet (specificity pocket) which offers favorable interaction. 

For chymotrypsin reaction pathway: what happens at step 2, given the blue boxes that precede and follow the step

2) ES Complex: First tetrahedral intermediate formed where Histidine acts as a base taking H+ from OH group of Ser (rate-determining step and the overall TS) Followed by, Serine195 oxygen performs nucleophilic attack on the carbonyl

• Oxyanion hole that stabilizes the tetrahedral intermediate.

“histidine takes H⁺ → serine attacks → tetrahedral intermediate forms.”

For chymotrypsin reaction pathway: what happens at step 3, given the blue boxes that precede and follow the step

3) Short-lived Intermediate: The first tetrahedral intermediate collapses, releasing product 1(via the help of the HIS being an acid and giving it's H+ to the departing N) and forming the acyl-enzyme intermediate.

For chymotrypsin reaction pathway: what happens at step 4, given the blue boxes that precede and follow the step

4) Acyl Enzyme intermediate(R-C=O-): Water molecule comes in the active site.

For chymotrypsin reaction pathway: what happens at step 5, given the blue boxes that precede and follow the step

5) Acyl Enzyme intermediate(R-C=O-): The water performs a nucleophilic attack on the acyl-enzyme intermediate & forms second tetrahedral intermediate via Histidine acting as a base by accepting H from water

• Oxyanion hole that stabilizes the tetrahedral intermediate.

For chymotrypsin reaction pathway: what happens at step 6, given the blue boxes that precede and follow the step

6) Short-lived Intermediate: The collapse of the tetrahedral intermediate, meaning the oxyanion hole is disrupted. Histidine acts as an acid(giving its H back to ser).

For chymotrypsin reaction pathway: what happens at step 7, given the blue boxes that precede and follow the step

7) EP Complex: Peptide product released from enzyme + enzyme regenerated

What group & amino acids form the oxyanion hole

Backbone amide N–H groups of Gly193 and Ser195.

In what steps are His acts as a base to form the tetrahedral intermediates

His acts as base in step 2&5 => forms tetrahedral intermediate (first forms-step 2, second forms-step 5)

In what steps are His acts as an acid to form Acyl enzyme intermediate

His acts as acid in step 3&6 => forms Acyl-enzyme intermediate (only form-step 3); step 3 release first product and step 6 is release of 2nd product

In which step does water perform nucleophilic attack on Acyl Enzyme intermediate

Step 5 water perform nucleophilic attack on Acyl-enzyme intermediate

In which step does ser perform nucleophilic attack

Step 2

For chymotrypsin reaction pathway, what is chymotrypsin

Chymotrypsin: the serine protease (e.g. elastase, trypsin, chymotrypsin, thrombin, plasmin) => break down protein

• A digestive enzyme produced in pancreas helping break down proteins in food to smaller peptides/amino acids

• Contributes to peptide bond cleavage that targets carboxyl end of amino acid

What are the catalytic triad of the chymotrypsin reaction pathway & other enzyme that have the triad as well

Histidine (57), Aspartate (102), and Serine (195) are catalytic triad at active site that lowers the G° of every step of catalysis that involves H+ transfer and TS binding

Asp positions His and helps stabilize its protonated form; His transfers protons; Ser attacks the substrate. Their precise arrangement makes catalysis possible.

serine protease (e.g. elastase, trypsin, chymotrypsin, thrombin, plasmin)

Understand the “limited chemistry” of amino acids (2 of them)

• Most electropositive atom

• Redox reactions reversibility

Amino acids have no more electropositive atoms than NH3+

Amino acids have little ability for reversible redox except for Cys-S-S-Cys disulfide, and this could cause enzyme structure to change

• For these and other reasons, enzymes use certain small molecules and ions.

Case example: Carboxypeptidase A needs Zn+2

Definitions of Non-AA components of Enzymes:

• Coenzyme

• Vitamin

• Metal ion

  • most abundant cofactor metal

Coenzyme: Organic compound required for enzymatic function. It is regenerated. (some: covalent bond while others: reversible enzyme binding)

Vitamin: Organic compound required in small amounts in the diet.

• i.e Vitamin B3 = Niacin = Nicotonic Acid

Metal ions: important roles as enzyme cofactors (~⅓ of all enzymes require metal ions)

• Maintain proper 3D structure

• Help bind substrate

• Participate in catalysis (e.g. redox)

  • iron or Fe2+

Categories of enzyme control

• Is enzyme there?

  • 4 ways of Regulation of synthesis (transcription, splicing, RNA degradation, translation)

Overview: Determination of RNA polymerase structure by Roger Kornberg’s laboratory work that won him Nobel Prize in 2006

4 ways of regulating/controlling protein synthesis

• Control transcription: Synthesize more or less mRNA

• Control splicing of mRNA: pre-mRNA enters as “RNA processing machine”, the spliceosome (most complex protein-RNA structure known: 5 small RNAs and ~200 different proteins bound together)

• Control of RNA degradation: RNA lifetime determined by degradation rate of mRNA molecule that is broken down into nucleotide fragments

• Regulation of protein synthesis on the ribosome (translation)

Categories of enzyme control

• Is enzyme there?

  • Regulation of degradation (e.g., ubiquitin-mediated proteasomal degradation)

    • Proteins lifetime(histone, Hb, Mitotic cyclin)

    • Principle

    • Mechanism in lysosome & cytosol

    • Why is it important to remove no longer needed protein

• Protein lifetimes (vary a lot): Histones ~ life of cell; Hb months; mitotic cyclin ~2 min

• Principles: Protein that partially unfolds or chemically reacts (e.g. oxidation) is recognized and then hydrolyzed. Healthy proteins can also be hydrolyzed!

  • Hydrolysis through fasting, infection, cell death (apoptosis) - Nobel Prize award in 2016 to Yoshinori Ohsumi for apoptosis discovery, cell cycle 

• Mechanisms:

  • a) In lysosome, proteins and whole organelles degrade by various proteases

  • b) In cytosol, the proteasome has the major role as the “protein degradation machine” consisting of 4 rows of 7 subunits 

    • Ubiquitin: 76 amino acid protein, found in all cells as marker for proteolysis 

• Why is it important to remove proteins no longer needed? 

  • Steps of cell division/DNA repair only when needed/destroy only defective newly synthesized proteins/immune activity turned off/transcription regulation (e.g. degradation of transcription repressor to turn on specific genes)

Categories of enzyme control

• Regulation of enzyme activity overview: R vs T state

Overview: Enzymes have two main states →

(1) active(R)

(2) less active meaning enzymes perform to have an “off-state” (T)

Categories of enzyme control

• Regulation of enzyme activity

  • Reversible(4) & Irreversible Covalent modification (many types, e.g. phosphorylation, proteolytic cleavage)

• Reversible through other enzymes, controlled by signals, via removal of chemical modification 

  • Addition of (a) phosphate group, (b) AMP, (C) Methyl group, (d) 100s of others

• Irreversible covalent modification 

  • Cleavage of peptide bond (proteolytic cleavage) through chymotrypsin

Categories of enzyme control

• Regulation of enzyme activity

  • Allosteric regulation (Example of allosteric control: ATCase[AKA Aspartate transcarbamoylase] (negatively regulated by CTP, positively regulated by ATP and why this makes sense, how these regulators shift V_in vs [S] curve; R T model)

• Allosteric Control: Binding at a site different from active site that is reversible between T and R transitions of quaternary structure + reversible allosteric regulation of monomer possible (e.g. human glucokinase) 

  • Covalent modification can also be allosteric (not mutually exclusive) 

• Key point: binds to another site and changes the shape of the active site, either activates or inhibits the protein function

• ATCase: first enzyme for characteristic identification of allosteric regulation 

  • ATP: Positive allosteric effector, favors R-state(Left)

  • Substrates (e.g. carbamoyl phosphate, and Asp) are also positive allosteric effectors

  • CTP: Negative allosteric effector, favors T-state (Right)

  • [S] a concentration unit like M

  • Vin a concentration unit / time

Be able to plot (or recognize) Vin vs [S] to show sigmoidal curve and impact of positive and negative allosteric regulators; where populations of R vs T state are high/low on this plot.

Key takeaway:

R state: looks like myoglobin curve

No allosteric effectors: somewhat of sigmoidal graph

Positive allosteric effectors: shifts the graph to the left

Negative allosteric effectors: shifts the graph to the right

T State: linear line(lowest activity)

They all have different Vmax