Presentation is loading. Please wait.

Presentation is loading. Please wait.

Enzymes How enzymes work?.

Published by Modified over 9 years ago

Similar presentations

Presentation on theme: "Enzymes How enzymes work?."— Presentation transcript:

1 Enzymes How enzymes work?

2 Tightly bound to Enz.  Prosthetic group
Enzymes Catalytic RNA (RNA as a catalyst, substrate is RNA) Transesterification and phosphodiester bond hydrolysis (cleavage) Self-splicing group I intron RNase P in E. coli Hammerhead ribozyme Catalytic antibody (abzyme) Ab generated with the transition-state analog as Ag Proteins (in their native conformations) Enzyme = Protein = Protein + cofactor (inorganic ions) = Protein + coenzyme (organic molecules) Tightly bound to Enz.  Prosthetic group Holoenzyme = Apoenzyme + cofactor/coenzyme Complete, catalytically active

3 Naming of enzymes Reactant + -ase
p. 192 Reactant + -ase 6 classes (Table 6.3), based on the reaction type Oxidoreductase, 氧化還原酶, (A- + B A + B+) Transferase, 轉移酶, (A–B + C A + B-C) Hydrolase, 水解酶, (A–B + H2O A-H + B-OH) X Y Lyase, 裂解酶, (A–B A=B + X-Y) X Y Y X Isomerase, 異構酶, (A-B A-B) Ligase, 接合酶, (synthetase) (A + B A-B)

4 Energy Diagram of a chemical reaction
Substrate (S) Product (P) Fig 8-2 Free energy change Affect equilibrium DG < 0, favor P Not a stable intermediate Activation energy

5 Enzymes lowers the activation energy
E + S ES EP E + P Reflect reaction rate do not change equilibrium Fig 8-3 ES EP ** DG*cat Intermediates, local min. energy The binding energy (DGB) released = Lowered DG* DGB: from multiple weak E-S interactions Catalysis and specificity

6 Catalytic power vs. Specificity
Enzyme-substrate interaction: “Lock and Key” hypothesis Enzymes are structurally complementary to their substrates. Induced-fit hypothesis A conformational change of E is induced by initial binding with S, which optimize the ES interaction. + E S ES + ES From Stryer

7 Enzyme kinetics S P, measure the initial rate (Vo) E Experiment:
[E]: fixed [S]: increasing Measure Vo = [P]/time E S1 S2 S3 … Fig 8-11 Michaelis-Menten equation At high [S], Vo = Vmax Maximum velocity At low [S], Vo  [S]

8 Kinetic model E + S ES E + P
[S], Vo, Vmax, and Km can be determined by exp. Michaelis-Menten kinetics Steady-state kinetics Before ES builds up: pre-steady state After [ES] reaches const. : steady state E + S ES k1 k-1 E + P k2 k-2 p. 259, (8-10) fast Slow  Rate limiting step

9 Michaelis-Menten kinetics
Vo = Vmax [S] Km + [S] Km = [S], when Vo = 1/2Vmax Fig 8-12 Km: Michaelis constant The conc. of substrate that will produce ½Vmax.

10 Lineweaver-Burk equation
Vo Vmax[S] Km Vmax 1 = + Plot 1/V vs. 1/[S] y-intercept: 1/Vmax x-intercept: -1/Km Slope: Km/Vmax 1/[S] 1/Vo 1/Vmax -1/Km Slope = Km/Vmax See Box 8-1 Double-reciprocal plot

11 Exercise A biochemist obtains the following set of data for an enzyme that is known to follow Michaelis-Menten kinetic. Please make a Michaelis-Menten plot. Please make a Lineweaver-Burk plot (double reciprocal plot). Vmax for the enzyme is ________. Km for the enzyme is _________. Substrate conc. Initial velocity [S], mM Vo (mmole/min) 1, 5,

12 kcat and kcat/Km E + S ES E + P k1 k-1 k2
kcat, rate constant or turnover number (轉換數) kcat (s-1) = Vmax/[Etotal] At saturation, kcat = k2, Vmax = kcat[Etotal] The limiting rate of any enzyme-catalyzed reaction at saturation. Enzyme efficiency: the number of S  P in a given unit of time when the E is saturated with S. Specificity constant: kcat/Km Used to compare different enzymes Upper limit: M-1s-1, diffusion-controlled

13 Second-order reaction (I)
A + B P + Q (bi-substrate) Single-displacement (sequential) reaction Ternary complex formation Both substrates must bind to the enzyme before any products are released The addition of A and B may be ordered or random, so is the release of products P and Q (Fig 8-13a, 8-14a) enz   A-enz-B  P-enz-Q   enz B   P  Q A  Compulsory order (Ordered Bi Bi) Random order (Random Bi Bi)

14 Second-order reaction (II)
A + B P + Q (bi-substrate) Double-displacement (ping-pong) reaction One substrate binds to the enzyme and one product is released before the second substrate binds (no ternary complex formed) (Fig 8-13b, 8-14b) A  B  enz  A-enz  P-enz  enz  B-enz  Q-enz  enz  P  Q

15 Example of a Ping-Pong reaction
1st step of amino acid catabolism in liver: transamination by aminotransferase (transaminase) B A Q P p. 628, Fig 18-4 enz  HN3+-enz  HN3+-enz  enz B   P  Q A 

16 Bisubstrate reactions
Fig 8-13, 8-14 Ternary complex formed

17 Enzyme and inhibitors Irreversible inhibition (p. 268)
Inhibitors bind and destroy the active sites e.g. Nerve gas (DIFP) and ACE ACE: acetylcholinesterase, catalyze the hydrolysis of acetylcholine (a neurotransmitter) Chymotrypsin (Fig 8-16) e.g. Asprin and prostaglandin synthetase Prostaglandin => pain … Suicide or mechanism-based inactivators Drug design Reversible inhibition (p. 266) Competitive Uncompetitive Mixed (non-competitive)

18 Competitive inhibition
Inhibitor (I) competes with S for the same active site on E to form EI I has similar structure as S Fig 8-15a

19 Competitive inhibition
In presence of a competitive inhibitor, [E] constant Vmax unchanged, Km increased [S] Km V Box 8-2, fig 1 + inhibitor aKm Vo = Vmax [S] aKm + [S] (8-28) Apparent Km (exp. determined)

20 Competitive inhibition
Medical application Methanol Formaldehyde Tissue damage e.g. blindness Alcohol dehydrogenase in liver Ethanol Acetaldehyde CH3CH2OH + NAD+  CH3CHO + NADH + H+ CH3COO- + NADH + 2H+ Acetaldehyde dehydrogenase Mitochondrial: low Km Cytosolic: high Km

21 Uncompetitive inhibition
Inhibitor (I) binds to a different site from S I binds ES complex to form ESI Box 8-2, fig 2 Fig 8-15b Both Km and Vmax decreased. Parallel lines

22 Mixed inhibition Inhibitor (I) binds a different site from S
I binds both E and ES Noncompetitive inhibition (a special case) Box 8-2, fig 3 Fig 8-15c

23 Non-competitive inhibition
In presence of a non-competitive inhibitor 1/V + inhibitor -1/Km 1/[S] A special case of mixed inhibition Km unchanged, Vmax decreased

24 Enzyme activity is affected by pH
Ionization state of key a.a. In the active site In structural recognition Fig 8-17 Stomach Hepatocyte

25 Steady-state vs. Pre-steady state
Before [ES] reaches constant Stryer 5th ed. Fig 8-13A

26 Pre-steady state kinetics
P-nitrophenylacetate hydrolysis by chymotrypsin Acylation – fast (initial burst) Deacylation – slow Fast step Slow step } initial burst Stoichiometric (E:S) p. 275, Fig 8-20

27 Hexokinase (p. 275-276) Inactive Active A bisubstrate enzyme
Induced fit Glucose vs. H2O Glucose vs. xylose Fig 8-21 Inactive Active D-glucose

28 Regulatory enzymes (I)
Allosteric enzyme Conformational change Does not follow M-M kinetics Non-covalent modification Homotropic: substrate = modulator, e.g. O2 binding of Hb Heterotropic: substrate  modulator e.g. feedback inhibition (Fig 6-??) E1 is an allosteric enzyme: S = Thr, M = Ile

29 Regulatory enzymes (II)
Covalent modification  all-or-none (Fig 6-30) Reversible e.g. phosphorylation/dephosphorylation (Fig 6-31) Fig 6-30 (1) Enz Enz— P — O- — O- O ATP ADP Inactive Active Pi

30 Regulatory enzymes (III)
Polypeptide cleavage (Fig 6-33) Inactive form  active form e.g. chymotrypsinogen  chymotrypsin e.g. trypsinogen  trypsin Inactive precursor: zymogen, proenzyme, proprotein Irreversible activation  inactivated by inhibitors Fig 6-33, right

Download ppt "Enzymes How enzymes work?."

Similar presentations

Enzymes and Coenzymes I Dr. Sumbul Fatma Clinical Chemistry Unit Department of Pathology.

Enzymes and Coenzymes I Dr. Sumbul Fatma Clinical Chemistry Unit Department of Pathology.
ENZYMES: KINETICS, INHIBITION, REGULATION

ENZYMES: KINETICS, INHIBITION, REGULATION
Enzyme Kinetic Zhi Hui.

Enzyme Kinetic Zhi Hui.
Lehninger Principles of Biochemistry

Lehninger Principles of Biochemistry
1 Enzymes Enzymes are biological catalysts. Recall that a catalyst speeds up the rate of a reaction by decreasing the activation energy needed for the.

1 Enzymes Enzymes are biological catalysts. Recall that a catalyst speeds up the rate of a reaction by decreasing the activation energy needed for the.
Enzyme Mechanisms.

Enzyme Mechanisms.
Enzyme Kinetics. Rate constant (k) measures how rapidly a rxn occurs AB + C k1k1 k -1 Rate (v, velocity) = (rate constant) (concentration of reactants)

Enzyme Kinetics. Rate constant (k) measures how rapidly a rxn occurs AB + C k1k1 k -1 Rate (v, velocity) = (rate constant) (concentration of reactants)
Enzymes. What is an enzyme? globular protein which functions as a biological catalyst, speeding up reaction rate by lowering activation energy without.

Enzymes. What is an enzyme? globular protein which functions as a biological catalyst, speeding up reaction rate by lowering activation energy without.
 The active site have a rigid shape.  Only substrates with the matching shape can fit.  The substrate is a key that fits the lock of the active site.

 The active site have a rigid shape.  Only substrates with the matching shape can fit.  The substrate is a key that fits the lock of the active site.
Chapter 8: Enzymes: Basic Concepts and Kinetics Copyright © 2007 by W. H. Freeman and Company Berg Tymoczko Stryer Biochemistry Sixth Edition.

Chapter 8: Enzymes: Basic Concepts and Kinetics Copyright © 2007 by W. H. Freeman and Company Berg Tymoczko Stryer Biochemistry Sixth Edition.
General Features of Enzymes Most biological reactions are catalyzed by enzymes Most enzymes are proteins Highly specific (in reaction & reactants) Involvement.

General Features of Enzymes Most biological reactions are catalyzed by enzymes Most enzymes are proteins Highly specific (in reaction & reactants) Involvement.
Enzymes Have properties shared by all catalysts Enhance the rates of both forward and reverse reactions so equilibrium is achieved more rapidly Position.

Enzymes Have properties shared by all catalysts Enhance the rates of both forward and reverse reactions so equilibrium is achieved more rapidly Position.
Chapter 5 (part 3) Enzyme Kinetics (cont.). Michaelis-Menton V max K m K cat K cat /K m E + S ESE + P k1k1 k -1 k cat Vo = Vmax [S] Km + [S] V max K m.

Chapter 5 (part 3) Enzyme Kinetics (cont.). Michaelis-Menton V max K m K cat K cat /K m E + S ESE + P k1k1 k -1 k cat Vo = Vmax [S] Km + [S] V max K m.
Chapter Twenty One Enzymes and Vitamins. Ch 21 | # 2 of 47 Catalysts for biological reactions Proteins Lower the activation energy Increase the rate of.

Chapter Twenty One Enzymes and Vitamins. Ch 21 | # 2 of 47 Catalysts for biological reactions Proteins Lower the activation energy Increase the rate of.
Enzyme Kinetics and Catalysis II 3/24/2003. Kinetics of Enzymes Enzymes follow zero order kinetics when substrate concentrations are high. Zero order.

Enzyme Kinetics and Catalysis II 3/24/2003. Kinetics of Enzymes Enzymes follow zero order kinetics when substrate concentrations are high. Zero order.
Chapter 13 Enzyme Kinetics

Chapter 13 Enzyme Kinetics
Enzymes. Definition of an enzyme Enzymeprotein Enzyme is protein catalystincrease the rate of reactions catalyst (i.e. increase the rate of reactions)

Enzymes. Definition of an enzyme Enzymeprotein Enzyme is protein catalystincrease the rate of reactions catalyst (i.e. increase the rate of reactions)
Enzyme activity is measured by the amount of product produced or the amount of substrate consumed. The rate of the enzymatic reaction is measured by the.

Enzyme activity is measured by the amount of product produced or the amount of substrate consumed. The rate of the enzymatic reaction is measured by the.
Enzyme Kinetics - Inhibition. Types of Inhibition Competitive Inhibition Noncompetitive Inhibition Uncompetitive Inhibition Irreversible Inhibition.

Enzyme Kinetics - Inhibition. Types of Inhibition Competitive Inhibition Noncompetitive Inhibition Uncompetitive Inhibition Irreversible Inhibition.
ENZYME KINETIC M. Saifur R, PhD. Course content  Enzymatic reaction  Rate of Enzyme-Catalyzed Reactions  Quatification of Substrate Concentration and.

ENZYME KINETIC M. Saifur R, PhD. Course content  Enzymatic reaction  Rate of Enzyme-Catalyzed Reactions  Quatification of Substrate Concentration and.

Similar presentations

Ads by Google