Ch 6: enzyme regulation

Hemophilia A:

- sex-linked trait - Deficiency in factor VIII

trypsin inhibitor:

- small protein made in pancreas - Trypsin inhibitor binds to any traces of active trypsin that might be present before it is secreted into the intestine. - It binds to the active site of trypsin, blocking its action but not itself being cut into tiny pieces.

phosphorylation---regulatory event:

-many enzymes and co-receptors are turned on or off phosphorylation and dephosphorylation -signals amplified in protein kinase cascades

4 ways enzyme activity can be regulated:

1. Allosteric 2. Reversible covalent modification 3. Binding of regulatory proteins 4. Irreversible proteolytic cleavage

Glycogen phosphorylase regulated by

1. Covalent modification- phosphorylation/dephosphorylation 2. Allosteric regulation: • Activators: cAMP • Inhibitors: ATP and Glucose-6-phosphate

Negative regulators: for coagulation: coagulation needs to be highly regulated, or vessels will get blocked

1. Serine protease - Protein C inactivates Va and VIIIa 2. Antithrombin III: serine protease inhibitor - Covalent bond formed between ATIII Arg and Ser of active site of thrombin and factor X.

Anti-coagulants:

1. Warfarin inhibits Vitamin K - Vitamin K is required for forming Ca2+ binding sites in VII, IX, X and prothrombin (Glu to g-carboxyglutamate modification) - allows the localization of coagulation factors to activated platelets. 2. Heparin increases ATIII affinity for Xa and thrombin 3. Drugs - Aspirin - inhibits cyclooxygenase needed for thromboxanes production. - less aggregation of platelets

Regulation can be:

1. noncovalent modification 2. covalent modification » irreversible » reversible

allosteric enzymes:

2 binding sites: 1) Active site for Ligand 2) Allosteric site for Modulator (allosteric modulator or allosteric effector) -when ligand modulator binds --> conformational changes which changes the property of the active site for the substrate. -if ligand mod.=substrate=homotropic -if mod doesn't =substrate=heterotropic

allosteric enzyme propeties:

Allosteric enzymes are generally multi-subunit enzymes. • Allosteric site and the active site are localized in separated subunits. --Binding of a ligand modulator to one subunit results in a conformational change which is transmitted to the other subunit containing the active site for the substrate molecule. (cooperativity)

Enzyme regulation by reversible covalent modification:

Involves post-translational modification of specific residues is common both in prokaryotes and eukaryotes. --phosphorylation, adenylation, uridylylation, ADPribosylation, methylation, acetylation and glycosylation.

allosteric enzymes to not exhibit:

Michaelis-Menton kinetic behavior --Homotropic allosteric enzymes have a sigmoid substratesaturation curve. --Heterotropic allosteric enzymes have substrate saturation curves of different shapes

non-covalent modification via allosteric enzymes:

Substrate concentration at which half the Vmax is achieved is called K0.5 -Substrate concentration giving 50% of velocity of allosteric enzyme catalyzed reaction --Homotropic allosteric enzymes have sigmoid substrate saturation curve

effect of ligand mod:

negative= will be less active positive= will be more active

Proteolysis of trypsin

• *Function:* hydrolyzes proteins into smaller peptides or amino acids in the intestine. • *Production*: Trypsin is produced in the pancreas in the form of inactive zymogen, trypsinogen. (in fig: enteropeptidase, not kinase)*** *secretion*: into the small intestine

enzyme act. by phosphorylation:

• Active p53 tumor suppressor/ transcription factor gene stimulates transcription of genes that suppress the cell cycle, even to the extent that the cell undergoes apoptosis. • However, this activity should be limited to situations where the cell is damaged or physiology is disturbed. • p53 protein is extensively regulated - >18 different phosphorylation sites. • Dephosphorylated p53 is inactive.

The blood coagulation cascade uses irreversible covalent modification:

• Blood coagulation in an important regulatory cascade. • Very sensitive response to injury • Amplification of molecular signal X → Y → Z → Fibrins along with platelets and RBCs forming blood clots

Enzyme regulation by reversible covalent modification examples:

• Dinitrogen reductase: involved in nitrogen fixation in bacteria. • Diptheria toxin: ADP-ribosylates and inactivates EF2. • Cholera toxin: ADP-ribosylates and inactivates G-proteins.

mechanism of proteolysis of trypsin:

• Enteropeptidase activates it into trypsin by proteolytic cleavage. • The resulting trypsins themselves activate more trypsinogens (autocatalysis), so only a small amount of enteropeptidase is necessary to start the reaction.

what are reg enzymes:

• Exhibit increased or decreased catalytic activity in response to certain signals. • Generally catalyze the rate limiting step in a metabolic pathway. • Allow the organism to adjust to change in environmental conditions and developmental stage.

glycogen breakdown:

• Glycogen breakdown increases with low glucose or energy level • Glycogen breakdown decreases with high glucose or energy. • Glycogen phosphorylase is the first enzyme in glycogen synthesis pathway.

feedback inhibition:

• In multi-enzyme metabolic pathways, usually the first enzyme is the allosteric enzyme. • The first enzyme is often inhibited by the end product by negative allosteric regulation.

Enzyme regulation by proteolytic cleavage:

• Many enzymes are synthesized as inactive precursor. • Activation of such enzymes involve proteolytic cleavage of the inactive precursor to produce fully active enzyme molecule. • If inactive precursor is that of protease, it is called *zymogen.* Chymotrypsinogen is a zymogen of chymotrypsin. Trypsinogen is a zymogen of trypsin. cleavage = irreversible.

Enzyme inactivation by phosphorylation:

• Phosphorylation-mediated enzyme inhibition in the tyrosine kinase called "src" (pronounced "sarc"). • When src is phosphorylated on a particular tyrosine, it folds on itself. • Thus masks its own kinase domain, and is shut "off".

Case I: Substrate-saturation curve of heterotropic allosteric enzymes.

• Positive modulation: K0.5 decreases without a change in Vmax • Negative modulation: K0.5 increases without a change in Vmax

Case II: Substrate-saturation curve of heterotropic allosteric enzymes. (less common)

• Positive modulation: Vmax increases without a change in K0.5 • Negative modulation: Vmax decreases without a change in K0.5

phosphorylation:

• Ser, Thr, Tyr are phosphorylated when ATP donates its γ-PO32- • His and Asp phosphorylation occurs in prokaryotes. --Phosphorylation imparts negative charge - conformational changes due to ionic interactions (with backbone N or Arg side chain) and H-bonding. --Adding a phosphate (PO4) to a polar R group of an amino acid can increase the polar and hydrophilic character of the protein.

Enzyme regulation by binding of inhibitors:

• Some enzymes are regulated by binding of inhibitor molecules. • Pancreatic trypsin inhibitor binds to and inhibits trypsin. • a1-antiproteinase inhibits neutrophil elastase, a protease acting on elastin (connective tissue).