BAHAN KULIAH BIOKIMIA POWER POINT BAGIAN 1 /BIOCHEMISTRY POWER POINT LECTURES PART 1 | Karya Tulis Ilmiah

Enzyme Catalysis

General Properties of Enzymes

•_{Increased reaction rates sometimes 10}6 to 1012 increase

Enzymes do not change G just the reaction rates. •Milder reaction conditions

•_{Great reaction specificity}

Substrate specificity

The non-covalent bonds and forces are maximized to bind substrates with considerable specificity

•Van der Waals forces

•electrostatic bonds (ionic interactions) •Hydrogen bonding

•Hydrophobic interaction

A + B P + Q

Substrates Products

 









NAD

CH

CH

NADH

H

OH

CH

CH

_{3 2 3}

O

Enzymes are Stereospecific

Yeast

N C H NH₂ O +

CH

₃

C

D

₂

OH +

N C H NH₂ O D

CH

₃

C-

D

O

+

Pro-

R hydrogen gets pulled off

Ox.

Red.

NADD

NAD

+

2. NAD

D

+ CH

₃

-C-H

O

C

H D

OH

CH₃

C H

D

OH

CH₃

3. CH3-C

-D

+ NADH

O

If the other enantiomer is used, the D is not transferred

Both the Re and Si faced transfers yield identical

products. However, most reactions that have an Keq for reduction >10-12 use the pro-R hydrogen while those

reactions with a Keq <10-10 use the pro-S hydrogen.

Specific residues help maintain

stereospecificity

Liver alcohol dehydrogenase makes a mistake

1 in 7 billion turnovers. Mutating Leu 182 to

Ala increases the mistake rate to 1 in 850,000.

This is a 8000 fold increase in the mistake rate,

Geometric specificity

Selective about identities of chemical groups but

Enzymes are generally not molecule specific

Similar shaped molecules can be highly toxic

N

C H

NH₂

O

+

N

N CH₃

Tobacco Nicotine

Because of this closeness in name

Coenzymes

Coenzymes: smaller molecules that aid in enzyme chemistry. Enzymes can:

a. Carry out acid-base reactions b. Transient covalent bonds

c. Charge-charge interactions Enzymes can not do:

d. Oxidation -Reduction reactions e. Carbon group transfers

Prosthetic group - permanently associated with an enzyme or transiently associated.

Commom Coenzymes

Coenzyme Reaction mediated

Biotin Carboxylation

Cobalamin (B12) Alkylation transfers

Coenzyme A Acyl transfers

Flavin Oxidation-Reduction

Lipoic acid Acyl transfers

Nicotinamide Oxidation-Reduction Pyridoxal Phosphate Amino group transfers

Vitamins are Coenzyme precursors

Vitamin Coenzyme Deficiency Disease Biotin Biocytin not observed Cobalamin (B₁₂) Cobalamin Pernicious anemia Folic acid tetrahydrofolate Neural tube defects

Megaloblastic anemia Nicotinamide Nicotinamide Pellagra

These are water soluble vitamins. The Fat soluble vitamins are vitamins A and D.

Humans can not synthesize these and relay on their

presence in our diets. Those who have an unbalanced diet may not be receiving a sufficient supply.

Niacin (niacinamide) deficiency leads to pellagra

Regulation of Enzymatic Activity

There are two general ways to control enzymatic activity. 1. Control the amount or availability of the enzyme. 2. Control or regulate the enzymes catalytic activity. Each topic can be subdivided into many different

categories.

Enzyme amounts in a cell depend upon the rate in which it is synthesized and the rate it is degraded. Synthesis rates can be transcriptionally or translationally controlled.

The catalytic activity of an enzyme can be altered either positively (increasing activity) or negatively (decreasing activity) through conformational

alterations or structural (covalent) modifications.

Examples already encountered is oxygen, carbon dioxide, or BPG binding to hemoglobin.

Also, substrate binding to the enzyme may also be modified by small molecule effectors changing its catalytic site.

Protein phosphorylation of Ser residues can activate or deactivate enzymes. These are generally

Aspartate Transcarbamoylase:

the first step in pyrimidine biosynthesis.

C O

NH₂

OPO₃

--CH₂ C C H O O -COO -H₃N+

CH₂ C C H O O -COO -N C O NH₂ H + + ATCase

H₂PO₄

-Carbamoyl Aspartate N--Carbamoyl aspartate phosphate

Notice the S shaped curve (pink) cooperative binding of aspartate

Positively homotropic cooperative binding

Hetertropically inhibited by CTP

Feedback inhibition

Where the product of a metabolic pathway inhibits is own

synthesis at the beginning or first

CTP is the product of this pathway and it is also a

precursor for the synthesis of DNA and RNA (nucleic acids). The rapid synthesis of DNA and/or RNA

depletes the CTP pool in the cell, causing CTP to be

released from ATCase and increasing its activity. When the activity of ATCase is greater than the need for CTP, CTP concentrations rise rapidly and rebinds to the

enzyme to inhibit the activity.

Enzymatic catalysis and mechanisms

•_{A. Acid - Base catalysis} •_{B. Covalent catalysis}

•_{C. Metal ion aided catalysis} •_{D. Electrostatic interactions}

•E. Orientation and Proximity effects

•_{F. Transition state binding} General Acid Base

Rate increase by partial proton abstraction by a Bronsted base or







D glucose



_k



_{D glucose}



obs  

 



  

dt d

v

Mutarotation of glucose by acid and base catalysts

K_obs = apparent first order kinetics but increases with increased concentrations of acid and base.

The acid HA donates a proton to ring oxygen, while the base abstracts a proton from the OH on carbon 1. To form the linear form. The cycle reverses itself after

This compound does not undergo mutarotation in aprotic solvents. Aprotic solvents have no acid or base groups i.e. Dimethyl sulfoxide or dimethyl formamide. Yet the reaction is catalyzed by

phenol, a weak acid and pyridine, a weak base

The reaction can be catalyzed by the addition of

-Pyridone as follows

v=k'[-pyridone][TM-a-D-glucose]

k' = 7000M x k or 1M -pyridone equals

Many biochemical reactions require acid

base catalysis

•Hydrolysis of peptides

•Reactions with Phosphate groups •Tautomerizations

•Additions to carboxyl groups

Asp, Glu, His, Cys, Tyr, and Lys have pK’s near

physiological pH and can assist in general acid-base catalysis.

Two histidine residues catalyze the

reaction. Residue His 12 is deprotonated and acts as a general base by abstracting a proton from the 2' OH.

His 119 is protonated and acts as a general acid catalysis by donating a proton to the phosphate group.

Covalent catalysis

Covalent catalysis involves the formation of a

transient covalent bond between the catalyst and

Catalysis has both an nucleophilic and an electrophilic stage 1 Nucleophilic reaction forms the covalent bond

Depending on the rate limiting step a covalent

catalytic reaction can be either elecrophilic or

nucleophilic. Decarboxylation by primary amines

are electrophilic because the nucleophilic step of

Schiff base formation is very fast.

Nucleophilicity is related to the basicity but

instead of abstracting a proton it attacks and forms

a covalent bond

.

Lysines are common in formation of schiff bases while thiols and imidazoles acids and hydroxyls also have

properties that make good covalent catalysts

Metal ion catalysts

One-third of all known enzymes needs metal ions to work!!

1. Metalloenzymes: contain tightly bound metal ions: I.e. Fe++, Fe+++, Cu++, Zn++, Mn++, or Co++.

2. Metal-activated enzymes- loosely bind ions Na+, K+, Mg+ +, or Ca++.

They participate in one of three ways:

a. They bind substrates to orient then for catalysis

b. Through redox reactions gain or loss of electrons. c. electrostatic stabilization or negative charge

Metal ions are effective

catalysts because unlike

protons the can be

present at higher

concentrations at

Metal ions can ionize water at higher

concentrations

The charge on a metal ion makes a bound water more acidic than free H2O and is a source of HO- ions even

below pH 7.0



_NH



_Co 



_{H O}



_



_NH



_Co3

 

_OH- _{ H}

5 3 2

3 5

3

Carbonic Anhydrase









H

O

HCO

H

Proximity and orientation effects





_k



_imidizole



_{p NO Ac}



_k



_{p NO Ac}



dt

O NO

p d

2 1

2 1

2 _{      }

k'

₁

= 0.0018s

-1

when [imidazole] = 1M

When the phenyl acetate form is used

k

₂

= 0.043 or 24k'

₁

•Reactants are about the same size as water molecules (approximation)

•Each species has 12 nearest neighbors (packed spheres) •Reactions only occur between molecules in contact

•Reactant conc. Is low so only one can be in contact at a time

B

A

B

A





 

k1





  





pairs 2

1 A B k A,B

k dt B A d    v





  

55.5M

B

A

12

B

A,

_pairs







_{pairs 1}





_pairs

1

55.5

12

A,

B

4.6k

A,

B

k

Only a 4.6 rate enhancement but molecular motions if slowed down leads to a decrease in entropy and rate enhancements.

Preferential transition state binding

Binding to the transition state with greater affinity to either the product or reactants.

RACK MECHANISM

Strain promotes faster rates

The strained reaction more closely resembles the transition state and interactions that preferentially

bind to the transition state will have faster rates

EP

ES

P

S

E N

k

k



 





 



kN for uncatalyzed reaction

and

EP

ES

ES

K

K

E

P

E

S

S

E

E N

K

T

R

K





 











 











‡ _‡ ‡ ‡

 

  

E

S

ES

K

_R







  

E S ES

K_T 

‡

‡













S

E

S

E



N

K

‡ ‡





 

ES

ES

K

_E‡



‡

 



  

N

E

 

S

k

T

 

S

T

K

 

S

k

v

N

N

B



N





























h

k

h

B





_{‡ ‡}



ES



k

T

K

 

ES

T

k

v

E

B

B



E

Preferential transition state binding

The more tightly an enzyme binds its reaction’s

transition state (K

_T

) relative to the substrate

(K

_R

) , the greater the rate of the catalyzed

reaction (k

_E

) relative to the uncatalyzed

reaction (k

_N

)

Catalysis results from the preferred binding





      



RT G G_{N E}

exp

k

k

N

E 10

6 rate enhancement

requires a 106 higher

affinity which is 34.2 kJ/mol

The enzyme binding of a transition state (ES‡ ) by two

hydrogen bonds that cannot form in the Michaelis Complex (ES) should result in a rate enhancement of 106 based on this