Enzyme regulation by –Allosteric control (include feedback inhibition) –Stimulation and inhibition by control proteins –Reversible covalent modification –Proteolytic activation Enzyme Regulation - Regulatory Strategies

Allosteric Control Example: Feedback Inhibition. Feedback inhibition –Seen in multi-enzyme systems –One enzyme acts as a regulatory enzyme –When end product exceeds cell’s requirement, it inhibits specifically the regulatory enzyme –All other enzymes in the system are slowed as a result of lowered substrate level. Example: E1 of the L-Ile biosynthesis (from L- Thr) enzyme system –E1: Threonine dehydratase, is specifically inhibited allosterically by L-Ile, the end of product, but not by any of the four intermediates –L-Ile binds at a regulatory not the active site

Activation of Protein Kinase A by cAMP (Allosteric Control) Protein Kinase A (PKA) –Alters activities of target proteins by phosphorylating specific Ser/Thr –Activated by cAMP –Mechanism of activation (allosteric) R 2 C 2 (in the absence of cAMP) –2 catalytic unit, high affinity –2 regulatory unit, high affinity for cAMP R 2 and 2C (in the presence of cAMP) R chain binds C chain –pseudosubstrate sequence (Arg-Arg-Gly-Ala-Ile) in R –Blocks active site of C cAMP binding –Induces conformational change in R –Causes dissociation of C from R (unblock active site)

Covalent Modification Some regulatory enzymes undergo reversible covalent modification –Modifying groups Phosphoryl (-PO 3 2- ) (on Tyr, Ser, Thr, His) adenylyl (Tyr) uridylyl (Tyr) adenosine diphosphate ribosyl (Arg, Gln, Cys, diphthamide - a modified His) methyl groups (Glu) –Covalently linked to and removed from the regulatory enzyme by separate enzymes

Enzyme Regulation by Phosphorylation Phosphorylation –Most common type of reversible covalent modification –Affects structure and therefore regulates activities of many enzymes –Phosphorylation carried out by protein kinases R-OH + ATP R-O-PO ADP + H + –Protein dephosphorylation carried out by protein phosphatases R-O-PO H 2 O R-OH + P i Residues that can be phosphorylated: Ser, Thr, Tyr

Effects of Phosphylation (Phosphoryl groups affect the structure and catalytic activity of proteins) Adds to negative charges  electrostatic interaction  structural changes  change in substrate binding or catalytic activity (e.g., repel negative charges on Glu/Asp or favorable interaction either electrostatic or H- bonding with Arg) Phosphoryl group capable of 3 H-bonds, highly directional Phosphorylation is fast  enzyme can be turned on/off fast Example: glycogen phosphorylase

Ser Glycogen Phosphorylase a and b differ in their secondary, tertiary, and quaternary structures The active site undergoes changes in structure and, consequently changes in catalytic activity as a consequence of phosphorylation /dephosphorylation How? N-term 20 amino acids (contains basic residues such as Arg) interact with acidic residues somewhere else. Phosphorylation of Ser14 disrupts these interaction and results in conformational change. Glycogen Phosphorylase More active Less active

Ser phosphorylation site (Yellow) Allosteric activator AMP (dark blue) Glucose (red) bound at active site Pyridoxal phosphate (PLP, light blue) (Vit B 6 derivative) Active site Subunit 1 Subunit 2 Phosphorylase a

Proteolytic Activation: Zymogen to Active Protease Activation of proteases –Pepsinogen (stomach) to pepsin –Trypsinogen (pancrease) to trypsin –Chymotrypsinogen (pancrease) to chymotrypsin –Procarboxypeptidase (pancrease) to carboxypeptidase –Proelastase (pancrease ) to elastase –Blood clotting enzymes Proinsulin (protein hormone) to insulin Procollagenase to collagenase Activation of zymogens in the control of developmental processes

Trypsinogen is the Common Activator of All the Pancreatic Zymogens Concurrent action of digestive proteases in duodenum Trypsin activates: trypsinogen chymotrypsinoge proelastase procarboxypeptidase What activates trypsinogen first to produce trypsin? Enteropeptidase (secreted by duodenum)

Chymotrypsinogen Activation slide 1 Chymotrypsinogen ( inactive)  chymotrypsin ( active) –chymotrypsinogen (245aa single chain) to  -chymotrypsin (active) by trypsin –  -chymotrypsin to  - chymotrypsin by chymotrypsin

Mechanism of Chymotrypsinogen Activation slide 2 Newly formed N-term (Ile16) (+ly charged) turns inward and interacts with Asp194 Induces conformational change Incomplete substrate binding site becomes complete Summary: Hydrolysis of a single peptide bond results in highly localized conformational changes that switches the enzymatic activity of the enzyme

Pancreatic Trypsin Inhibitor Zymogen (inactive)  active enzyme –Proteolysis (irreversible) Active enzyme  inactive enzyme –Protease inhibitors Pancreatic trypsin inhibitor (6 kDa) –K d = 0.1 pM (tight binding to trypsin) –Complex cannot be dissociated with 8 M urea or 6 M guianidine –A very effective substrate analog –Almost perfectly complementary to active site

 1-antitrypsin and Pulmonary Emphysema  1-antitrypsin (  1-antiproteinase) (plasma protein) –Protects tissue from digestion by elastase ( secreted by neutrophils, white blood cells that engulf bacteria ) –Blocks elastase much better than trypsin  1-antitrypsin deficiency and emphysema –Genetic disorders lead to  1-antitrypsin deficiency –Excess elastase digests elastic fibers (elastin & collagen type IV) and other connective tissue proteins –This destroys alveolar walls in the lungs  emphysema Cigarette smoking increases the likelihood of develop emphysema –Smoke oxidizes Met358 of the inhibitor, essential for elastase binding

Pepsinogen Activation Pepsin (digest proteins in the highly acidic environment of the stomach) –pH optimum 2 –Catalytic residues: 2 Asp –First 44 amino acids removed proteolytically and spontaneously below pH 5 Mechanism of activation –Highly basic precursor segment (6 Arg&Lys, + charged) –Highly acidic pepsin moiety (including catalytic Asp residues) –Fully formed active site is blocked in zymogen form –Lowering pH below 5 disrupts salt bridges and exposes active site