Principles of Biochemistry 5 | Enzyme Catalysis| Class Notes |HarvardX

Enzyme Catalysis

© Gerlt J A. 1994

Though the spontaneous reaction could happen automatically, it could run very slow without the help of the catalyzes.

Why Enzyme Can Catalysis

• Transient covalent bonds
  • Activate substrate, lower the activate-energy…
• Weak non-covalent interaction
  • binding energy: $ \Delta G_B$ (Figure: Enzymatic Reaction, D), needed energy to decrease the reaction
• Desolvation: weak interaction between water and substrate is replaced by weak interaction between the substrate and the enzyme.

Catalysis is driven by structure and conformational changes.

© HarvardX

Exp: Beta-galactosidase

$\beta-glycosidic$ bond
$\alpha-glycosidic$ bond

$\beta-galactosidase$ can only hydrolysis the $\beta-glycosidic$ bond but not
$\alpha-glycosidic$ bond though they are highly similar.

Why enzyme

Size of the enzyme

Compared to the small active site, the enzyme is relatively large.
Role of the outside residues:

• position the active residues in the right position
• optimizing catalysis by induced fit
• enzyme regulation:
  • Methylation
  • phosphorylation: tyrosine, serine, and threonine residues.
• enzyme complexes
• conformation changes after binding the substrate
  short peptide:
• few conformation possibilities.

3-point attachment model

Phenomenon: In ethanol, two hydrogen bonds to the Carbon, and both of them are equally expected to be transferred to NADH， but only one of the hydrogen could be transferred.

In this model, the enzyme attaches 3 positions of ethanol:

• methyl group
• hydroxyl group
• one specific hydrogen from the methylene group

Protein Processing

DNA -> mRNA -> Protein ->
Post translation modification:

• Substrate Dependence
• Allosteric Control
• Covalent Modification: Phosphorylation
• Proteolytic: from zymogen and proenzyme to functional protein by proteolytic enzyme digestion.

Proteolytic

The fate of the protein

The different fate of protein translation:

• Translated freely in the cytoplasm by free ribosomes
• Transported into organisms once been translated
• Ribosomes were soon attached to the ER and peptides went to the Golgi transport system to Plasma membrane or lysosomes.

Enzyme Chymotrypsin

Chymotrypsin:

• a zymogen
• a digested enzyme created by the pancreas
• Started with an inactive form: chymotrypsinogen.
• work in the Duodenum to digest the food.
• 245 residues

1. chymotrypsinogen + Trypsin -> 15 residues peptide + 230 residues peptide
2. $\Pi-Chymotrypsin$:

• [1:15] S-S [16:245] (disulfide bond)

3. Autolysis:

• [1:13] S-S [16-146] S-S [149:245]

4. the mature form of chymotrypsin: $\alpha-Chymotrypsin$

Insert an model:
PDBID:

Hormone: Insulin

Insulin:

• secreted by the pancreas;
• regulating the level of blood glucose;

1. Rre-pro-insulin: 110 residues
2. Pro-insulin
3. Folding: intramolecular disulfide bonds link together the N-terminal and the C-terminal
4. Golgi
5. Digested to insulin + C-peptide
6. Secreted

insulin: A (N-terminal)+ B (C-terminal)

C-Protein:
Previously Recognition: It’s for structure support and inactivity by-product.
Recent research: involved in many physiological events.

• opposing with insulin in red blood cells

Case 1: Revers Regulation

In red blood cell: PDE3 + ATP -> AMP

When PED3 is deactivated: ATP is released, enzyme ENOS is working:

• ENOS + ATP + NO(nitric oxide) -> Vasodilation

C-Protein +[binding]+ GPR146 (receptors) -> PKC -> prohibited PDE3

Case 2: Both have inhibitory effects.

WBC (White blood cell):
release cytokines-> active adhesion molecules of the endothelial cells -> WBC attach to the endothelial cell -> extravasation
WBC binds both C-protein and insulin: decreases the release of cytokines:
Prevent tissue infiltration to reduce inflammation.

Case 3: C-Protein mediates the tuning of insulin signaling.

• Low insulin levels:
  • C-peptide binds to the membrane receptor
  • Activate G-protein
  • Insulin signaling enhanced
• High insulin level:
  • C-peptide binds the membrane receptor
  • Activate another G-protein
  • Insulin signaling repressed

Conclusion:
Protein processing is an important way to regulate protein activity:

• Stored as precursors to avoid toxicity
• Quick response to acute change with active precursors.

digraph F { rankdir ="LR" subgraph A { rank = same node [style=filled, color = green]; DNA mRNA Protein } subgraph cluster_0 { rank = same node [style=filled, font=Courier, color = red, shape = box]; sub1 [label = "Substrate Dependence"] sub2 [label = "Allosteric Control"] sub3 [label = "Covalent Modification"] sub4 [label = "Proteolytic", shape = "", color = green ] style=filled; color=lightgrey; node [style=filled,color=white]; label = "Post Translation Modification"; } DNA -> DNA [label="Replication"] DNA -> mRNA [label = "Transcription"] mRNA -> Protein [label = "Translation"] Protein -> sub1 Protein -> sub2 Protein -> sub3 Protein -> sub4 }digraph F { rankdir ="LR" ribosome1 [label="Free Ribosome"] ribosome2 [label ="ER Ribosome"] PM [label = "Plasma Membrane"] Mitochondrial [label = "Mitochondrial"] subgraph A { rank = same Mitochondrial Preoxisome Nuclear Lysosomes Secretory PM } subgraph B { rank = same } ｍRNA -> ribosome1 -> Cytoplasm ribosome1 -> Mitochondrial ribosome1 -> Preoxisome ribosome1 -> Nuclear ｍRNA -> ribosome2 ribosome2 -> ER -> Golgi -> PM ER -> Lysosomes ER -> Secretory }digraph F { node [style=filled, color = green, shape = box]; P1 [label = "1-245" ] subgraph cluster_1{ subgraph B{ rank = same P3_1 [label = "1-13"] P3_2 [label = "16-146"] P3_3 [label = "149-245"] } label = "Autolysis: alpha-Chymotrypsin" } subgraph cluster_0 { subgraph A{ rank = same P2_1 [label = "1-15"] P2_2 [label = "16-245"] } P2_1 P2_2 label ="Trypsin: pi-Chymotrypsin" } P1 -> P2_1 P1 -> P2_2 P2_1 -> P3_1 P2_2 -> P3_2 P2_2 -> P3_3 P2_1 -> P2_2 [label ="disulfide" arrowhead = "none", color = "red"] P3_1 -> P3_2 [label ="disulfide" arrowhead = "none", color = "red"] P3_2 -> P3_3 [label ="disulfide" arrowhead = "none", color = "red"] }digraph F{ node [shape = box] Pre [label = "Pre-pro-insulin"] Pro [label = "Pro-insulin"] subgraph A{ rank = "same" Pro ProP [label = "Pro-insulin"] } ProC [label = "C-terminal"] ProN [label = "N-terminal"] subgraph B { rank = "same" } Pre -> Pro [label="remove part of AAs (Signal peptide)"] Pro -> ProP [label="folding"] ProN -> ProC [ label ="disulfide", color = "red", arrowhead="none"] ProN->ProP->ProC [arrowhead="none"] subgraph cluster_0{ CP insulin style=filled; color=lightgrey; node [style=filled,color=white]; label = "Golgi: Final Product"; } CP [label="C-protein"] Out [label="Out Cell"] ProP -> CP ProC -> insulin ProN -> insulin CP -> Out [label="secrete"] insulin -> Out }digraph F{ rankdir = "UD" CP [label = "C-Protein", style = "filled", color ="green"] insulin [style = "filled", color ="red"] subgraph cluster_0{ subgraph A { rank = same marr0001 PDE3 } "marr0001" [shape=diamond,style=filled,label="",height=.1,width=.1] ; "insulinR" [label = "insulin receptor"] ATP -> marr0001 [dir=none,weight=1] marr0001 -> AMP PDE3 -> marr0001 [label="hydrolysis"] GPR146 -> PDE3 [label ="Prohibited"] insulinR -> PDE3 [label ="Activate" arrowhead = "tee"] label ="Red Blood Cell" color = "pink" style=filled; } subgraph cluster_1{ subgraph AA{ rank = "same" marr0002 marr0003 } "marr0002" [shape=diamond,style=filled,label="",height=.1,width=.1] ; "marr0003" [shape=diamond,style=filled,label="",height=.1,width=.1] ; ENOS NO -> marr0003 [dir = "none"] marr0003 -> Vasodilation label = "Endothelial Cell" } CP -> GPR146 insulin -> insulinR ENOS -> marr0002 [arrowhead = "none"] ATP -> marr0002 [arrowhead = "none"] marr0002 -> marr0003 }digraph F { "marr0001" [shape=diamond,style=filled,label="",height=.1,width=.1] ; "marr0002" [shape=diamond,style=filled,label="",height=.1,width=.1] ; subgraph A { rank = same insulin marr0001 marr0002 } subgraph B { rank = same CP cytokines } WB [label ="White Blood Cell", shape="circle"] CP [label = "C-Protein", shape = "box", style = "filled", color = "green"] insulin [shape = "box", style = "filled", color="deeppink1"] infiltration [shape ="invhouse", color = "cadetblue1", style = "filled"] WB -> marr0002 -> cytokines -> infiltration CP -> marr0001 insulin -> marr0001 marr0001 -> marr0002 [label="Prohibit"] }digraph{ node [shape = "box"] Hi [label = "High insulin level"] Li [label = "Low insulin level"] Is [label = "insulin signaling" ] subgraph A { rank = same CP1 [label = "C-Protine"] "marr0001" [shape="point",style=filled,label="",height=.1,width=.1] ; "marr0002" [shape=point,style=filled,label="",height=.1,width=.1] ; } Hi -> marr0001 -> Receptor2 -> GP2 [ color ="green"] Li -> marr0002 -> Receptor1 -> GP1 [ color ="red"] marr0001 -> CP1 -> marr0002 [dir=none] GP1 -> Is [label="active", color ="red"] GP2 -> Is [label="prohibit", color = "green"] }