Enzymes

Enzymes Jason Ryan, MD, MPH Www.Medicalstudyzone.com.

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Enzymatic Reactions S E P S + E ⇄ ES ⇄ E + P Www.Medicalstudyzone.com.

Enzymatic Reactions Image courtesy of Wikipedia/U+003F S + E ⇄ ES ⇄ E + P Www.Medicalstudyzone.com.

Michaelis-Menten Kinetics V [S] V = Reaction velocity Rate of P formation Vmax V = Vm* [S] Km + [S] Www.Medicalstudyzone.com.

Michaelis-Menten Kinetics • Adding S → More P formation → Faster V • Eventually, reach Vmax Www.Medicalstudyzone.com.

Michaelis-Menten Kinetics • At Vmax, enzymes saturated (doing all they can) • Only way to increase Vmax is to add enzyme Www.Medicalstudyzone.com.

Enzyme Kinetics [S] Vmax Vmax More Enzyme Www.Medicalstudyzone.com.

Michaelis-Menten Kinetics V [S] V = Reaction velocity Rate of P formation Vmax V = Vm* [S] Km + [S] Www.Medicalstudyzone.com.

Michaelis Constant (Km) V = Vm * [S] Km + [S] Key Points: 1. Km has same units as [S] 2. At some point on graph, Km must equal [S] Www.Medicalstudyzone.com.

[S] + [S] 2 [S] 2 Michaelis Constant (Km) V = Vm * [S] = Vm * [S] = Vm When V = Vm/2 [S] = Km Www.Medicalstudyzone.com.

[S] Vmax V = Vm* [S] Km + [S] Michaelis Constant (Km) Vmax/2 Km Www.Medicalstudyzone.com.

V = Vm* [S] Km + [S] • Small Km → Vm reached at low concentration [S] • Large Km → Vm reached at high concentration [S] Michaelis Constant (Km) [S] Vmax Vmax/2 Km Www.Medicalstudyzone.com.

V = Vm* [S] Km + [S] Michaelis Constant (Km) • Small Km → Substrate binds easily at low [S] • High affinity substrate for enzyme • Large Km → Low affinity substrate for enzyme [S] Vmax Vmax/2 Km Www.Medicalstudyzone.com.

Key Points • Km is characteristic of each substrate/enzyme • Vm depends on amount of enzyme present • Can determine Vm/Km from • Michaelis Menten plot V vs. [S] • Lineweaver Burk plot 1/V vs. 1/[S] Www.Medicalstudyzone.com.

Lineweaver Burk Plot V = Vm* [S] Km + [S] 1 = Km + [S] = Km + [S] V Vm [S] Vm [S] Vm[S] 1 = C * 1 + 1 V [S] Vm Www.Medicalstudyzone.com.

Lineweaver Burk Plot 1 V 1 S 1 Vm -1 Km Km Vm Www.Medicalstudyzone.com.

Enzyme Inhibitors Jason Ryan, MD, MPH Www.Medicalstudyzone.com.

Enzyme Inhibitors • Many drugs work through enzyme inhibition • Two types of inhibitors: • Competitive • Non-competitive Www.Medicalstudyzone.com.

Enzymatic Reactions S E P S + E ⇄ ES ⇄ E + P Www.Medicalstudyzone.com.

Enzyme Inhibitors S E I Competitive Competes for same site as S Lots of S will overcome this S E P Non-competitive Binds different site S Changes S binding site S cannot overcome this Effect similar to no enzyme I Www.Medicalstudyzone.com.

Competitive Inhibitor [S] Vmax Vmax/2 Km Normal Km Same Vm Higher Km Inhibitor Www.Medicalstudyzone.com.

Non-competitive Inhibitor [S] Vmax Vmax With inhibitor Vmax/2 Vmax/2 Lower Vm Same Km Km Www.Medicalstudyzone.com.

Competitive Inhibitor 1 V 1 S 1 Vm -1 Km Normal Www.Medicalstudyzone.com.

Competitive Inhibitor 1 V 1 S 1 Vm -1 Km -1 Km Normal Inhibitor Www.Medicalstudyzone.com.

Non-competitive Inhibitor 1 V 1 S 1 Vm -1 Km Normal Inhibitor 1 Vm Www.Medicalstudyzone.com.

Inhibitors Competitive • Similar to S • Bind active site • Overcome by more S • Vm unchanged • Km higher Non-competitive • Different from S • Bind different site • Cannot be overcome • Vm decreased • Km unchanged Www.Medicalstudyzone.com.

Dose-Response Jason Ryan, MD, MPH Www.Medicalstudyzone.com.

Efficacy • Maximal effect a drug can produce • Morphine is more efficacious than aspirin for pain control Www.Medicalstudyzone.com.

Potency • Amount of drug needed for given effect • Drug A produces effect with 5mg • Drug B produces same effect with 50mg • Drug A is 10x more potent than drug B • More potent not necessarily superior • Low potency only bad if dose is so high it’s hard to administer Www.Medicalstudyzone.com.

Pain Control Analgesia Dose (mg) Morphine Aspirin Www.Medicalstudyzone.com.

Dose-Response • For many drugs we can measure response as we increase the dose • Can plot dose (x-axis) versus response (y-axis) Www.Medicalstudyzone.com.

Dose-Response • Graded or quantal responses • Graded response • Example: Blood pressure • Can measure “graded” effect with different dosages • Quantal response • Drug produces therapeutic effect: Yes/No • Example: Number of patients achieving SBP<140mmHg • Can measure “quantal” effect by % patients responding to dose Www.Medicalstudyzone.com.

Graded Dose Response Curve Dose Effect Emax E50 10 20 30 40 50 60 Www.Medicalstudyzone.com.

Graded Dose Response Curve Log [Dose] Effect Emax E50 1 10 100 ↓EC50 = ↑Potency Www.Medicalstudyzone.com.

Graded Dose Response Curve Log [Dose] Effect Emax E50 EC50/Potency A > B > C A B C Potency Www.Medicalstudyzone.com.

Graded Dose Response Curve Log [Dose] Effect Emax E50 EMax/Efficacy B>A A B Efficacy Emax Www.Medicalstudyzone.com.

Competitive Antagonists Log [Dose] Effect Emax E50 Receptor Agonist Receptor Agonist + Competitive Antagonist Www.Medicalstudyzone.com.

Non-competitive Antagonists Log [Dose] Effect Emax E50 Receptor Agonist Receptor Agonist + Non-Competitive Antagonist E50 EC50 Max Effect Www.Medicalstudyzone.com.

Spare Receptors • “Spare” receptors: Activate when others blocked • Maximal response can occur even in setting of blocked receptors • Experimentally, spare receptors demonstrated by using irreversible antagonists • Prevents binding of agonist to portion of receptors • High concentrations of agonist still produce max response Www.Medicalstudyzone.com.

Spare Receptors Log [Dose] Effect Agonist + Low Dose Non-Competitive Antagonist Agonist + High Dose Non-Competitive Antagonist Emax Source: Basic and Clinical Pharmacology, Katzung Www.Medicalstudyzone.com.

Partial Agonists • Similar structure to agonists • Produce less than full effect Www.Medicalstudyzone.com.

Partial Agonists Agonist or Partial Agonist Given Alone Log [Dose] Effect Emax Full Agonist Partial Agonist Max Effect Effect similar to agonist plus NC antagonist Www.Medicalstudyzone.com.

Partial Agonist Single Dose Agonist With Increasing Partial Agonist Log [Dose Partial Agonist] % Binding 100% Agonist Partial Agonist 0% Www.Medicalstudyzone.com.

Partial Agonist Single Dose Agonist With Increasing Partial Agonist Log [Dose Partial Agonist] Response 100% Agonist Response Partial Agonist Response 0% Total Response Www.Medicalstudyzone.com.

Partial Agonists • Pindolol/Acebutolol • Old antihypertensives • Activate beta receptors but to less degree that norepinephrine • “Intrinsic sympathomimetic activity” (IMA) • Lower BP in hypertensive patients • Can cause angina through vasoconstriction • Buprenorphine • Partial mu-opioid agonist • Treatment of opioid dependence • Clomiphene • Partial agonist of estrogen receptors hypothalamus • Blocks (-) feedback; ↑LH/FSH • Infertility/PCOS Www.Medicalstudyzone.com.

Quantal Dose Response Curve Log [Dose] % Patients Therapeutic Response Adverse Response 100% 50% ED50 LD50/ TD50 Www.Medicalstudyzone.com.

Therapeutic Index • Measurement of drug safety Therapeutic Index = LD50 ED50 Www.Medicalstudyzone.com.

Therapeutic Window Therapeutic Window Log [Dose] % Patients 100% 50% LD50 Minimum Effective Dose Minimum Toxic Dose Www.Medicalstudyzone.com.

Low TI Drugs • Often require measurement of levels to avoid toxicity • Warfarin • Digoxin • Lithium • Theophylline Www.Medicalstudyzone.com.