Enzyme Phosphorylation Aulanni’am Biochemistry Laboratory Chemistry Department Brawijaya University Aulani " Biokimia Enzim" Presentasi 11

Aulani " Biokimia Enzim" Presentasi 11 Enzyme regulation: How to control the activity of a pathway in response to the needs of the cell or body Aulani " Biokimia Enzim" Presentasi 11

Multiple ways of controlling enzyme activity Control of enzyme availability Amount of enzyme in the cell Control of enzyme synthesis Control of enzyme degradation I could add examples as I go along for these situations. E.g, start out now saying there is tissue-specific expression of the gene for phosphorylase, etc. (instead of waiting until the last slide). Location of enzyme in the cell On/off membrane Inside/outside organelle Aulani " Biokimia Enzim" Presentasi 11

Multiple ways of controlling enzyme activity Control of enzyme activity Allosteric activation or inactivation: interaction of small molecule with enzyme, but not at active site Aulani " Biokimia Enzim" Presentasi 11

Aulani " Biokimia Enzim" Presentasi 11 “Feedback” control of metabolic pathways is often accomplished by allosteric mechanisms 1 2 3 4 5 6 A B C D E F G (Excess G) Aulani " Biokimia Enzim" Presentasi 11

Aulani " Biokimia Enzim" Presentasi 11 Allosteric modulation of aspartate transcarbamylase, the first unique step in the biosynthesis of pyrimidines (C, T, U) Carbamoyl phosphate + aspartate ATCase N-carbamoyl aspartate CTP ATP Aulani " Biokimia Enzim" Presentasi 11

The reaction catalyzed by aspartate transcarbamoylase carbamoyl phosphate H2PO4- N-carbamoyl aspartate Aulani " Biokimia Enzim" Presentasi 11

Aulani " Biokimia Enzim" Presentasi 11 Activity of aspartate transcarbamoylase (and many allosteric enzymes is cooperative with respect to aspartate [aspartate] v0 Sigmoid (S-shaped) curve Aulani " Biokimia Enzim" Presentasi 11

Cooperativity in enzymatic activity Multimeric enzymes Binding of S to one protomer causes change in adjacent protomer, increasing the affinity of both protomers for the substrate Aulani " Biokimia Enzim" Presentasi 11

Cooperativity of enzymatic activity: multiple states of the enzyme ELA EMA (T state) Less active Lower affinity for S (R state) More active Higher affinity for S Aulani " Biokimia Enzim" Presentasi 11

What good is cooperativity in enzymatic activity? (Same Vmax and v0 at 1/2 Vmax) Steeper slope ensures greater sensitivity to variations in substrate concentration Aulani " Biokimia Enzim" Presentasi 11

Effects of positive and negative modulators on cooperative enzymes ATP and CTP are positive and negative modulators of aspartate transcarbamoylase [substate] v0 positive negative Aulani " Biokimia Enzim" Presentasi 11

Aulani " Biokimia Enzim" Presentasi 11 Allosteric activators and inhibitors stabilize different states of the enzyme (ATCase) ELA EMA (T state) Less active Lower affinity for S Stabilized by CTP (R state) More active Higher affinity for S Stabilized by ATP Aulani " Biokimia Enzim" Presentasi 11

Aulani " Biokimia Enzim" Presentasi 11 Allostery is accomplished by conformational changes in ATCase and other enzymes Red: upper catalytic trimer Blue: lower catalytic trimer Green: regulatory dimers (bind ATP and CTP) Aulani " Biokimia Enzim" Presentasi 11

Other about allosterism Allosteric modulators can change Km or Vmax or both Not all allosteric enzymes are cooperative Allosteric modulation renders an enzyme sensitive to metabolites other than its own substrates and products (e.g., ATCase is sensitive to endproducts of purine and pyrimidine biosynthesis). Aulani " Biokimia Enzim" Presentasi 11

Multiple ways of controlling enzyme activity Control of enzyme activity Allosteric activation or inactivation: interaction of small molecule with enzyme, but not at active site Interaction with a large molecule, usually another protein Example: Thrombin and thrombomodulin I said it’ not clear if you call this allosteric or not. Aulani " Biokimia Enzim" Presentasi 11

Modification of thrombin activity by thrombomodulin Thrombomodulin (on cell surface of vascular endothelium) Decreases activity of thrombin toward fibrinogen Increases ability of thrombin to activate Protein C, an inhibitor of clotting Aulani " Biokimia Enzim" Presentasi 11

> Modification of thrombin activity by thrombomodulin clot Thrombin Protein C Thrombomodulin Protein C zymogen (contains Gla) Aulani " Biokimia Enzim" Presentasi 11

Multiple ways of controlling enzyme activity Control of enzyme activity Allosteric activation or inactivation Interaction with another protein Reversible covalent modification Example: phosphorylation Aulani " Biokimia Enzim" Presentasi 11

Modulation by phosphorylation kinase ATP ADP phosphatase H2O Pi PO32- Enzyme X (active) Enzyme X (inactive) Phosphorylation may also result in inactivation, depending on the enzyme Aulani " Biokimia Enzim" Presentasi 11

Multiple ways of controlling enzyme activity Control of enzyme activity Allosteric activation or inactivation Interaction with another protein Reversible covalent modification Irreversible modification Example: zymogen activation Any other cool examples of irreversible modification? Maybe a ubiquitin-dependent degradation? Aulani " Biokimia Enzim" Presentasi 11

Multiple ways of controlling a single enzyme: Glycogen phosphorylase Gene expression Multiple genes, tissue-specific expression Rates transcription vary Protein activity Allosteric control Reversible phosphorylation Aulani " Biokimia Enzim" Presentasi 11

Aulani " Biokimia Enzim" Presentasi 11 to be continued . Aulani " Biokimia Enzim" Presentasi 11