Enzymes 1 Flashcards
What are enzymes ? What are ribozymes
Catalyst
A substance which increases the rate of a chemical reaction but remains unchanged at the end of the reaction
- Enzymes are biological catalysts
- Most enzymes are proteins (names end with ..ase)
- But some RNA (ribonucleic acid) molecules also show enzyme activity- ribozymes
What are enzymes essential for? If there is a deficiency of enzyme, what can it lead to? Enzyme defect can be due to? (3)
Enzymes essential for
- Breakdown of nutrients to supply energy & building blocks
- Assembling building blocks into proteins, nucleic acids, membranes, cells & tissues
Deficiency of enzyme activity can cause disease
Enzyme defects can be due to
üGenetic mutations (eg; phenylketonuria)
üNutritional deficits (eg; beri beri)
üViral or bacterial pathogens (eg; cholera toxin)
•
Testing for enzyme in blood can determine …? What other ways are enzymes used? Give example
- Testing for enzyme activities in blood & tissue fluids can help in diagnosis & prognosis of disease (eg; ALT for hepatitis)
- Enzyme inhibitors are used as drugs (eg; allopurinol for gout)
- Enzymes are employed in the synthesis of drugs
- Enzymes are used as therapeutic agents
- Rennin (a protease) used in the production of cheese
- Enzymes used as reagents in specific lab tests for biochemical analytes
•What can free energy change ( delta G) be used to predict? If (G) is negative..? If (G) is positive..?
- Change in free energy during a reaction (DG), can be used to predict the direction of a reaction
- Eg: A to B and B to A
- If delta G is negative; net loss of energy, exergonic; reaction goes spontaneously from A to B
- If delta G is positive; net gain of energy, endergonic; reaction does not go spontaneously from B to A
•DG depend on what? If the reaction goes forward, how is it written compared to backward reaction? When is DG =0? Are reactions with large negative DG reversible? How can a reaction go to completion?
- G depends on the concentration of reactants & products, therefore is variable
- DG of the forward reaction is equal in magnitude but opposite in sign to the back reaction
- Eg: if DG (forward) = – 5 kcal/mol; back reaction will have DG = + 5 kcal/mol
- When the reaction reaches equilibrium, no further net chemical change occurs, DG = 0
- Reactions with large negative DG are irreversible under physiologic conditions
- A reaction with +ve DG can be coupled to another reaction with a large –ve DG to make it go to completion (usually coupled ATP hydrolysis which releases energy)
What is the effect of enzyme on activation energy of a reaction?
∆G- change in free energy
- Indicates nothing about the rate of the reaction.
- No difference in the free energy change of the overall reaction between the catalyzed and uncatalyzed reaction.
How do enzymes work?
What is Ea?
What is the transition state?
How are uncatalyzed reactions in comparison to catalyzed?
- All chemical reactions have an energy barrier separating the reactants & products (Free energy of activation, Ea)
- Ea – energy difference between that of the reactants and transition state
- Transition state – a high-energy intermediate that occurs during formation of product
- Uncatalyzed chemical reactions are slow
- Molecules must possess enough energy to achieve the transition state
What doe an enzyme do in a reaction?
An enzyme
•Does not alter the free energy of reactants or products
•Does not change the equilibrium of the reaction
•Provides an alternate reaction pathway with a lower free energy of activation
•Lowers the free energy of activation – transition state is achieved faster
•Stabilizes the transition state
•Therefore accelerates the rate of reaction
Enzyme catalyzed reactions 103 – 108 times faster than uncatalyzed reactions
What is an enzyme’s active site? What does it contain? How does the enzyme accelerate the product formation?
- A site (a pocket or cleft) in the enzyme where the reaction takes place
- Contains a.a side chains that create a 3-dimensional surface complementary to the substrate
- Binds the substrate in a manner resembling the transition state of the molecule
- Provides catalytic groups that enhance the formation of transition state
- Stabilizes the substrate in its transition state; accelerates product formation
- E+S ® ES ® EP ® E + P
- Substrate binding causes a conformational change in the enzyme ® Induced fit ® Catalysis
What are the types of enzyme specificity? ( 2 groups and 4 sub groups)
Enzymes are highly specific interacting with one or a few substrates and catalyzing only one type of reaction
Types of enzyme specificity
1.Reaction specificity
Enzymes are classified into 6 groups based on the type of reaction they catalyze
2. Substrate specificity
** a. absolute specificity**
Some enzymes are absolutely specific for a single substrate
Eg: glucose oxidase acts only on glucose
b. Group specificity
Some enzymes act on a few related susbtrates
Eg: trypsin hydrolyzes peptide bonds formed by lysine and arginine
c. Stereospecificity
Enzymes are specific for the D- & L- isomers
Eg: D- & L- amino acid oxidases
d. Geometric isomer specificity
Enzymes show specificity for cis- or trans- isomers
Eg: fumarase acts on fumarate (trans) but not on maleate (cis)
How are most enzymes named? Give example of substrate name, description of reaction and trival names. How are systematic names given/ by what system? And how are they classified?
•Common names
–Most enzymes have suffix –ase attached to
•Substrate’s name Ex: Lactase, amylase, maltase
•Description of the reaction Ex: Lactate dehydrogenase
•Trivial names Ex: Trypsin, pepsin
•Systematic names
–By International Union of Biochemistry and Molecular Biology (IUBMB)
–All enzymes are classified into 6 major classes
–Classes are divided into subclasses and sub subclasses.
–Name of the enzyme consists of names of all the substrates; followed by the description of reaction, to which suffix –ase is added
–Ex: Lactate:NAD+ oxidoreductase
– Unambiguous and informative
Give the six groups of classification of enzymes
Based on the type of reaction they catalyze
1.Oxidoreductases
Catalyze oxidation-reduction reactions (eg: lactate dehydrogenase)
2. Transferase
Catalyze transfer of groups containing C-, N- or P- (eg: hexokinase)
3. Hydrolases
Catalyze cleavage of bonds by addition of water (eg: lactase)
4. Lyases
Catalyze cleavage of C-C, C-S & C-N bonds by mechanisms other than hydrolysis (eg: aldolase)
5. Isomerases
Catalyze interconversion of isomers (eg: phosphohexose isomerase)
6. Ligases
Catalyze formation of bonds between two molecules coupled to the hydrolysis of high-energy phosphate (ATP)
(eg: pyruvate carboxylase)
What are cofactors , coenzymes , holoenzymes and apoenzymes? Most of the co enzymes are derived from? What are cosubstrates and prosthethic groups?
•Some enzymes require non-protein additional factors for activity
•Holoenzyme (active enzyme) = Apoenzyme (protein part) + non-protein component
non-protein component = Organic-coenzymes +Inorganic- Cofactor (Metal ions)
- Most of the coenzymes are derived from B-complex vitamins
- Cosubstrates (transiently associated coenzymes) Eg- NAD+
- Prosthetic group (permanently associated coenzyme)Eg- FAD
For the following coenzyme name the vitamin it is derived from, the group transferred and dependent enzyme
TPP,FMN, FAD, NAD+,NADP+, LIPOIC ACID, PLP, CoA,FH-4,BIOTIN, COBALAMIN
What are the cofactor( metal ion ) for the following enzyme:
Carbonic anhydrase, Alcohol dehydrogenase, Carboxy peptidase
Hexokinase, G-6-phosphatase
Enolase
Phosphogluco mutase, Enolase
Dipeptidase, Superoxide dismutase
Salivary amylase
Tyrosinase, SOD
Cytochrome oxidase, Catalase, Peroxidase
Lipase
Xanthine oxidase
Glutathione peroxidase
What is kinetics? What is the rate or velocity of an enzymatic reaction? How is the rate expressed? Name the factors affecting the rate of an enzymatic reaction.
•Kinetics is a study of the rate of conversion of reactants to products
Rate or velocity of a enzymatic reaction (v)
- The number of substrate molecules converted to product per unit time under defined conditions
- Expressed as mmoles product formed/min
- International Unit (IU): amount of enzyme that catalyzes conversion of 1 mmole of substrate to product per minute
Factors affecting rate of an enzymatic reaction
a. Temperature
b. pH
c. Substrate concentration
Explain how temperature increases the rate of the reaction . What is the optimal temperature for most enzymes? What happens at higher temperatures?
- Velocity of an enzyme-catalyzed reaction inc with inc in temperature up to a maximum, and then declines
- With inc in temperature, inc collision of molecules, more molecules have sufficient energy to pass over the energy barrier
- Optimum temperature for most enzymes between 35-40°C
- At higher temperatures (usually above 40°C) most enzymes start losing their native structure (denaturation), reaction velocity slows down
At, higher temperaturesm most enzymes become inactive, except which of the following?
** (exceptions: enzymes from thermophilic bacteria, eg; Taq polymerase from T. aquaticus, used in PCR)**
Explain how pH affect the rate of reaction
Concentration of H+ ions influences enzyme activity by
- Affecting the state of ionization of chemical groups in the enzyme active site & substrate
- Causing denaturation of enzymes at extremes of pH
Velocity against pH graph: bell-shaped curve
Enzymes vary in their optimum pH preferences
How does the effect of substrate concentration affect the rate of reaction
- Rate of an enzyme-catalyzed reaction increases with increase in substrate concentration until a maximal velocity (Vmax) is reached
- At high substrate concentrations, all available binding sites on the enzyme are saturated with substrate
- Most enzymes show hyperbolic curve when velocity is plotted against substrate concentration [S] (Michaelis-Menten kinetics)
How does the michaelis-Menten kinetics reaction look like?
Compare first order reaction and zero order reaction
First order reaction: when [S] is much less than Km, reaction velocity is directly proportional to substrate concentration
Zero order reaction: When [S] is much greater than Km, reaction velocity independent of substrate concentration
Reaction velocity is directly proportional to enzyme concentration
What is Km?
Substrate concentration [S] at which vo is half of Vmax
- Is characteristic of an enzyme for its substrate
- Reflects the affinity of the enzyme for its substrate
- Does not vary with enzyme concentration
What is the significance of low Km and high Km?
Low Km:
- Reflects a high affinity of enzyme for its substrate
- Low concentration of substrate is needed to half-saturate the enzyme
High Km:
- Reflects a low affinity of enzyme for its substrate
- High concentration of substrate is needed to half-saturate the enzyme
Explain glucokinase and hexokinase in terms of which one has the higher km vs. lower km and which one has the higher Vmax vs. lower Vmax. When are they active?
Glucokinase and hexokinase
- Both phosphorylate glucose in glycolysis
- Glucokinase: higher Km for glucose (10 mmol/L)
- Hexokinase: lower Km for glucose (0.2 mmol/L)
- Glucokinase: higher Vmax
- Hexokinase: lower Vmax
- Hexokinase active even at low glucose level
- Glucokinase active after a meal
Give the mathematical relationship between the initial reaction velocity (Vo) and [S]
What is the Lineweaver-Burk plot ? What is the other name for it? What is in the x-axis and y-axis? And what is it used to calculate?
MM plot – not easy to determine Vmax accurately
- Linear form of MM plot; taking reciprocal of MM equation
- Also called double-reciprocal plot
- Plots the reciprocals of vo against reciprocals of [S]
- Used to calculate Km and Vmax accurately & to determine mechanism of action of enzyme inhibitors
