Enzyme Phosphorylation Aulanniam Biochemistry Laboratory Chemistry Department Brawijaya
Enzyme Phosphorylation Aulanni’am Biochemistry Laboratory Chemistry Department Brawijaya University 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 –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
“Feedback” control of metabolic pathways is often accomplished by allosteric mechanisms A 1 B 2 C 3 D 4 E (Excess G) Aulani " Biokimia Enzim" Presentasi 11 5 F 6 G
Allosteric modulation of aspartate transcarbamylase, the first unique step in the biosynthesis of pyrimidines (C, T, U) Carbamoyl phosphate ATCase N-carbamoyl + aspartate ATP Aulani " Biokimia Enzim" Presentasi 11 CTP
The reaction catalyzed by aspartate transcarbamoylase aspartate carbamoyl phosphate H 2 PO 4 - N-carbamoyl aspartate Aulani " Biokimia Enzim" Presentasi 11
Activity of aspartate transcarbamoylase (and many allosteric enzymes is cooperative with respect to aspartate v 0 Sigmoid (Sshaped) curve [aspartate] 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 (T state) Less active Lower affinity for S EMA (R state) More active Higher affinity for S Aulani " Biokimia Enzim" Presentasi 11
What good is cooperativity in enzymatic activity? (Same Vmax and v 0 at 1/2 Vmax) v 0 [S] 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 positive v 0 negative [substate] Aulani " Biokimia Enzim" Presentasi 11
Allosteric activators and inhibitors stabilize different states of the enzyme (ATCase) ELA (T state) Less active Lower affinity for S Stabilized by CTP EMA (R state) More active Higher affinity for S Stabilized by ATP 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 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 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 > Thrombin Protein C Thrombomodulin Protein C zymogen (contains Gla) Aulani " Biokimia Enzim" Presentasi 11 clot
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 ATP kinase ADP PO 32 - Enzyme X (inactive) phosphatase H 2 O Pi Enzyme X (active) 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 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
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