Lecture 9

Lecture 9 Introduction to enzyme

Sample questions for the nucleic acid section

Nucleoside is a pyrimidine or purine base

• A.covalently bonded to a sugar• B.ionically bonded to a sugar• C.hydrogen bonded to a sugar• D.none of the above

The sugar in RNA is ______ , the sugar in DNA is _____

• A.deoxyribose, ribose• B.ribose, deoxyribose• C.ribose, phosphate• D.ribose, uracil

Sample questions for the nucleic acid section

In gel electrophoresis, what fragments will move most quickly through a gel?

• A.Large fragments• B.Small fragments• C.Large genome• D.None of these

Nucleotide bases and aromatic amino acids absorb light respectively at

• A.280 and 260 nm• B.260 and 280 nm• C.270 and 280 nm• D.260 and 270 nm

Sample questions for the nucleic acid section

Which of the following is found on RNA but not DNA?

• A.Uracil• B.Deoxyribose• C.Phosphate• D.Adenine

Which is true about the pairing of bases in the DNA molecule?

• A. purines always pair with pyrimidines

• B. a single ring base pairs with another single ring base

• C. a double ring base pairs with another double ring base

• D. purines pair with purines and pyrimidines with pyrimidines

Sample questions for the nucleic acid section

A messenger acid is 336 nucleotides long, including the initiator and termination codons. The maximum number of amino acids in the protein translated from this mRNA is:

• A 999• B 630• C 330• D 111• E 110

With what mRNA codon would the tRNA in the diagram be able to form a codon-anticodon base pairing interaction?

• A. 3'-AUG-5'• B. 3'-GUA-5'• C. 3'-CAU-5'• D. 3'-UAC-5'• E. 3'-UAG-5'

Sample questions for the nucleic acid section

• Of what units are nucleic acids constituted? What are the chemical entities that compose that unit?

• What is the rule for the pairing of nitrogenous bases in the DNA molecule? And in the RNA?

• For each of the following structures identify: the carbohydrate (ribose or deoxyribose)?; nucleoside or a nucleotide? DNA or a RNA system?

Enzymes

• substance that increase rates of a chemical reaction

• does not effect equilibrium• remain unchanged in overall process

• reactants bind to catalyst, products are released

Lock & Key – Fischer (1894)A proposal for ES

Induced Fit – Koshland (1963)A proposal for ES

Enzymes increase product formation by (1) lowering the energy barrier (activation energy) for the product to form (2) increases the favorable orientation of reactant molecules for product formation to be successful (stabilize transition state intermediate)

• Activation Energy (AE) – The energy require to reach transition state from ground state.

• AE barrier must be exceeded for reaction to proceed.

• Lower AE barrier, the more stable the transition state (TS)

• The higher [TS], the move likely the reaction will proceed.

Enzymatic Catalysis

S Ts P

Catalytic Power

• Enzymes can accelerate reactions as much as 1016 over uncatalyzed rates!

• Urease is a good example: – Catalyzed rate: 3x104/sec

– Uncatalyzed rate: 3x10 -10/sec

– Ratio is 1x1014 !

Specificity

• Enzymes selectively recognize proper substrates over other molecules

• Enzymes produce products in very high yields - often much greater than 95%

• Specificity is controlled by structure - the unique fit of substrate with enzyme controls the selectivity for substrate and the product yield

Classes of enzymes

1. Oxidoreductases = catalyze oxidation-reduction reactions

2. Transferases = catalyze transfer of functional groups from one molecule to another.

3. Hydrolases = catalyze hydrolytic cleavage

4. Lyases = catalyze removal of a group from or addition of a group to a double bond, or other cleavages involving electron rearrangement.

5. Isomerases = catalyze intramolecular rearrangement.

6. Ligases = catalyze reactions in which two molecules are joined.

Enzymes named for the substrates and type of reaction

Co-enzymes

• Non-protein molecules that help enzymes function

• Associate with active site of enzyme• Enzyme + Co-enzyme =

holoenzyme (conjugated enzyme )• Enzyme alone = apoenzyme• Organic co-enzymes – thiamin, riboflavin, nia

cin, biotin• Inorganic co-enzymes – Mg ++, Fe++, Zn++, Mn++

Increase in the hydrogen ion concentration(pH) considerably influences the enzyme activity

Sample questions

What is the function of enzymes within living systems? • A) structural elements • B) neurotransmitters • C) catalysts • D) hormones

Enzymes have names that • A) always end in -ase • B) always end in -in • C) can end either in -in or -ase • D) can end in either -in or -ogen

Sample questions

The protein portion of a conjugated enzyme is called a(n) • A) apoenzyme. • B) coenzyme. • C) holoenzyme. • D) cofactor.

Which of the following could be a component of a conjugated enzyme? • A) coenzyme • B) cofactor • C) apoenzyme • D) more than one correct response • E) no correct response

Sample questions

Enzyme cofactors that bind covalently at the active site of an enzyme are referred to as _________.

• (a) cosubstrates. • (b) prosthetic groups. • (c) apoenzymes. • (d) vitamins

Sample questions

Which of the following statements concerning the effect of temperature change on an enzyme-catalyzed reaction is correct?

• A) An increase in temperature can stop the reaction by denaturing the enzyme.

• B) An increase in temperature can increase the reaction rate by increasing the speed at

• which molecules move. • C) An increase in temperature to the optimum temperature maximiz

es reaction rate. • D) more than one correct response • E) no correct response

Mechanisms of Enzyme Action

Transition (TS) State Intermediate

• Transition state = unstable high-energy intermediate• Rate of reaction depends on the frequency at which reac

tants collide and form the TS • Reactants must be in the correct orientation and collide

with sufficient energy to form TS• Bonds are in the process of being formed and broken in

TS• Short lived (10–14 to 10-13 secs)

Intermediates• Intermediates are stabl

e.

• In reactions with intermediates, 2 TS’s are involved.

• The slowest step (rate determining) has the highest activation energy barrier.

• Formation of intermediate is the slowest step.

•Enzyme binding of substrates decrease activation energy by increasing the initial ground state (brings reactants into correct orientation)

•Need to stabilize TS to lower activation energy barrier.

The Transition State

Understand the difference between G and G‡

• The overall free energy change for a reaction is related to the equilibrium constant

• The free energy of activation for a reaction is related to the rate constant

Reaction Coordinate

Sample questions• A catalyst can promote product formation during a chemical reaction

by _____. • (a) lowering the activation energy barrier. • (b) stabilizing the transition state. • (c) positioning reactants in the correct orientation. • (d) bringing reactants together. • (e) all of the above

Which of the following is characteristic of an enzyme catalyst? • (a) It positions reactants in the correct orientation. • (b) It lowers the activation energy barrier. • (c) It binds the transition state tighter than the substrate. • (d) all of the above

ES complex must not be too stable

Raising the energy of ES will increase the catalyzed rate

•This is accomplished by loss of entropy due to formation of ES and destabilization of ES by

•strain

•distortion

•desolvation

Transition State Stabilization

Transition state analog

• Equilibrium between ES <-> TS, enzyme drives equilibrium towards TS

• Enzyme binds more tightly to TS than substrate

Mechanistic Strategies

Active Sites

• The active site of an enzyme represents as the small region at which the substrate (s) binds and participates in the catalysis

• The active site is made up of amino acids, known as catalytic residues

Polar AA Residues in Active Sites

Sample questions

An enzyme active site is the location in the enzyme where • A) protein side groups are brought together by bending and folding t

o form a site for interactions with substrates • B) the catalyst interactions with the enzyme • C) catalyst molecules are generated • D) the substrate creates the catalyst molecules

An enzyme active site is the location in an enzyme where substrate molecules

• A) are generated. • B) become catalysts. • C) undergo change. • D) more than one correct response • E) no correct response

Common types of enzymatic mechanisms

• Substitutions reactions

• Bond cleavage reactions

• Redox reactions

• Acid base catalysis

• Covalent catalysis

Substitution Rxns

• Nucleophillic Substitution–

• Direct Substitution

C

O

XR

Y

C

O

XR

Y

C

O

YR+ X

C

R1 R2

R3 Y

CX YR1 R2

R3X

CR1 R2

X R3

+ Y

transition state

Nucleophillic = e- rich Electrophillic = e- poor

Oxidation reduction (Redox) reactions

• Loose e- = oxidation (LEO)

• Gain e- = reduction (GER)

• Central to energy production

• If something oxidized something must be reduced (reducing agent donates e- to oxidizing agent)

• Oxidations = removal of hydrogen or addition of oxygen or removal of e-

• In biological systems reducing agent is usually a co-factor (NADH of NADPH)

• Heterolytic vs homolytic cleavage• Carbanion formation (retains both e-)

R3-C-H R3-C:- + H+

• Carbocation formation (lose both e-) R3-C-H R3-C+ + H:-

• Free radical formation (lose single e-)R1-O-O-R2 R1-O* + *O-R2

Cleavage reactions

Hydride ion

• Accelerates reaction by catalytic transfer of a proton• Involves AA residues that can accept a proton• Can remove proton from –OH, -NH, -CH, or –XH• Creates a strong nucleophillic reactant (i.e. X:-)

Acid-Base Catalysis

X H : B X: H B

X H : B X: H B

C

O

N

O

H H : B

C

O

OH

N

H

B

C

O

OH HN

: B

:

:

Acid-Base Catalysis

carbanion intermediate

Covalent Catalysis• 20% of all enzymes employ covalent catalysis

A-X + B + E <-> BX + E + A

• A group from a substrate binds covalently to enzyme

(A-X + E <-> A + X-E)

• The intermediate enzyme substrate complex (A-X) then donates the group (X) to a second substrate (B) (B + X-E <-> B-X + E)

Covalent Catalysis

Protein Kinases

ATP + E + Protein <-> ADP + E + Protein-P

1) A-P-P-P(ATP) + E-OH <-> A-P-P (ADP) + E-O-PO4-

2) E-O-PO4- + Protein-OH <-> E + Protein-O- PO4

-

The Serine ProteasesTrypsin, chymotrypsin, elastase, thrombin, subtilisi

n, plasmin, TPA• All involve a serine in catalysis - thus the name • Ser is part of a "catalytic triad" of Ser, His, Asp • Serine proteases are homologous, but locations

of the three crucial residues differ somewhat • Substrate specificity determined by binding pock

et

A zymogen (or proenzyme) is an inactive enzyme precursor.

Serine Proteases are structurally Similar

Chymotrpsin Trypsin Elastase

Serine protease catalytic triad - from chymotrypsin

Substrate binding specificity

The serine proteases differ in their sequence and in their substrate specificity

Serine proteases 

Nucleophilic attack Protonation

the carbonyl oxygen becomes an oxyanion

The main chain NHs of Gly193 and Ser195 stabilize the negatively charged oxyanion of the tetrahedral intermediate

The reaction pathway for serine protease peptide bond cleavage

The highest energy peak in the serine protease reaction pathway corresponds to the covalent intermediate hydrolysis process, which is the slowest step in the reaction.