Nature ands Origin of Lifelecture 8

Demistifying Enzymes(mostly borrowed)

Law of Mass Action• The law of mass action is universal, applicable under any

circumstance. We introduce the mass action law by using a general chemical reaction equation in which reactants A and B react to give product C and D, in units of molecules per unit volume.

• a A + b B --> c C + d D….where a, b, c, d are the digital coefficients for a balanced chemical equation.

e.g. Fe2O3 + 3CO -----> 2Fe + 3CO2 • The mass action law states that if the system is at equilibrium

at a given temperature, then the following ratio is a constant.• [C]c [D]d = Keq [A]a[B]b

• The square brackets "[ ]" around the chemical species represent their concentrations. This is the ideal law of chemical equilibrium or law of mass action.

But for organic chemicals

Equilibrium is very slow.One frequently finds timescales for decay of hours,

days, months or years.As a result, there can be huge effects from speeding

up some organic chemical reactions, but not others.

In particular, if the product of a chemical reaction is rapidly removed, the equilibrium is effectively shifted in the direction of producing and using it.

An Enzyme is an evolved Catalyst,But what is a catalyst?

A catalyst is an ingredient that is used in a reaction, but is unchanged at the end.

It is a way of going around a hill instead of over the top!

Reactions are limited by activation energy, the repulsion that needs to be overcome to bring reactants together. Such energy occurs in the “tail” of a distribution.

But by dividing a reaction into a number of steps, each with a lower activation energy, the reaction can be speeded up.

Activation Energy

Enzyme action

OR the reverse direction

Terms1. Anabolic reactions:

2. Catabolic reactions:

3. Metabolism:

4. Catalyst:

5. Metabolic pathway:

6. Specificity:

7. Substrate:

8. Product:

Reactions that build up molecules

Reactions that break down molecules

Combination of anabolic and catabolic reactions

Sequence of {enzyme controlled} reactions

Only able to catalyse specific reactions

The molecule(s) the enzyme works on

Molecule(s) produced by enzymes

A substance that speeds up reactions without changing the produced substances

Enzymes lower activation energy by forming an enzyme/substrate complex

Substrate + Enzyme

Enzyme/substrate complex

Enzyme/product complex

Product + Enzyme

In anabolic reactions enzymes bring the substrate molecules together.

In catabolic reactions the enzyme active site affects the bonds in substrates so they are easier to break.

How?

• Enzymes catalyze reactions by stabilizing and vibrating about transition states.

• Enzymes are highly specific in both the reaction catalyzed, and the choice of reactants (both called substrates.)

However, some enzymes are focused on a specific molecular link, and ignore side chains, while others are specific to both the link and the total compound.

Equilibria and Inhibition

• Enzymes cannot alter the equilibrium of a chemical reaction. They accelerate the forward and backward reaction by the same factor.

• Enzymes may be inhibited by the growth in abundance of the final product. In this way the enzyme becomes active when the final product is needed.

• In addition, regulatory proteins can either stimulate or inhibit reactions.

RateEnzymes can increase rate by a factor of between 108

to 1026.

How is this done?1) Lower the reaction energy barrier by deforming the

molecule.2) Increase the contact time by using Van der Waals

forces to hold molecules together3) Increase the frequency of interaction, and the

number of attempts to cross the energy barrier, through Brownian vibration.

Frequency

• Suppose reactants need to be spaced correctly to 1A, (10-10m) and move at a velocity of 0.1Km/sec.

• Then the timescale of a “meeting” is 10-12sec.

So what might an enzyme do in a millisecond?

Can increase the timescale of contact by 109.Can properly orient the two substrates

(interactors).If the energy barrier is lowered by a factor 3,

there is a potential of an exp (2E/kT) improvement in the probability of an interaction.

Overall factors of improvements of billions or more are understandable.

Separation

• Presumably the energy output associated with the direction of the reaction, is used to break the bonds between the substrate(s) and the enzyme after the reaction.

Enzymes are globular proteins

• Active site has a specific shape due to tertiary structure of protein.

• A change in shape of the protein affects shape of active site and the function of the enzyme.

Characteristics of enzymes

• Only change the rate of reaction. They do not change the equilibrium or end products.

• Specific to one particular reaction

• Present in very small amounts due to high molecular activity:

Turnover number = number of substrate molecules transformed per minute by one enzyme molecule

Catalase turnover number = 6 x106/min

pH affects the formation of hydrogen bonds and sulphur bridges in proteins and so affects shape.

pepsintrypsin cholinesterase

2 4 8 106pH

Rate

of R

eacti

on (M

)

Many enzymes denature at ~ 60oC

Temperature

Rate

of r

eacti

on Rate doubles every 10oC

Enzyme denaturing and losing catalytic abilities

Optimum temperature

Some thermophilic bacteria have enzymes with optimum temperatures of 85oC or more.

Dependence on temperature• The 10 C law is inexact. • Reactions observed always have an

appreciable energy barrier, but its range is limited because we actually (1)observe the reaction and (2) its rate is not very fast.

• These results, together with the typical reaction temperature of 300K, result in a change in temperature of ~1 part in 30 giving a frequency of energy in the tail from a Gaussian distribution changing by about a factor 2 for this small temperature change.

Active sites 1

• The active site(s) of an enzyme are the regions that bind the substrate.

• The active site takes up a small part of the enzyme volume.

• The active site is a 3-D structure that comes from different parts of the amino acid sequence. E.g. in lysozyme, the amino acids are #s 35,52,62,63 and 101 in the 129 residue protein.

Active sites 2

Substrates are bound to enzymes by multiple weak (Van der Walls) interactions.

Active sites are clefts or crevices. Water is usually excluded unless it is a reactant.

Though the substrate fits the enzyme like a lock and key, the enzyme and substrate are flexible and change their shapes on contact.

Timeline of enzyme discovery1835:

Breakdown of starch to sugar by malt

1877:Name enzyme coined to describe chemicals in yeast that ferment sugars

1897:Eduard Buchner extracted enzyme from yeast and showed it could work outside cells

1926:James B Sumner produced first pure crystalline enzyme (urease)

and showed enzymes were proteins

1905:Otto Rohm exyracted pancreatic proteases to supply enzymes for tanning

1930-1936:Protein nature of enzymes finally established when digestive enzymes

crystallised by John H Northrop

1946: Sumner finally awarded Nobel prize

Ribozymes

• A ribozyme is an RNA molecule with a well defined tertiary structure that enables it to assist or perform a chemical reaction. Many ribozymes are catalytic, but some such as self-cleaving ribozymes are consumed by their reactions. Ribozyme means ribonucleic acid enzyme.

• It may also be called an RNA enzyme or catalytic RNA.

Origin of life speculations

What follows is not conventional thinking, but an attempt to understand issues by thinking “out of the box”

RNA First Catalyst??Quote From Wikipedia:• It had been a firmly established belief in biology

that catalysis was reserved for proteins. In retrospect, catalytic RNA makes a lot of sense. This is based on the old question regarding the origin of life: Which comes first, enzymes that do the work of the cell or nucleic acids that carry the information required to produce the enzymes? Ribo-Nucleic acids as catalysts circumvents this problem. RNA, in essence can be both the chicken and the egg.

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PROBLEM: RNA is a made-up molecule. • What selected the ingredients of RNA? • What catalyzed production of the first RNA?

The first catalyst problem1) The development needed to be driven by

survival selection.2) What survival use was the first RNA?3) What selected the ingredients of the first RNA?4) What catalyzed production of the first RNA? 5) What form was the first RNA? E.g. was it as a

tri-phosphate or a mono-phosphate? ( one has a role of producing condensation reactions and the other is an information molecule.)

6) What was the development sequence that produced RNA?

So-called Central Dogma (from Crick)This is just a working hypothesis!!

• Transfer of genetic information from protein to nucleic acid never occurs and never occurred???

• The original postulate that genetic information can be transferred only from nucleic acid to nucleic acid and from nucleic acid to protein,

• that is from DNA to DNA from DNA to RNA and from RNA to protein

• (although information transfer from RNA to DNA was not excluded and is now known to occur [reverse transcription]).

Options to explore

1) Could a sugar have been a first catalyst to assist inorganic phosphate?

2) If so, what selected bases?3) Could the Central Dogma stage have been

preceded by a stage in which amino acids came first and selected nucleic acids?

4) Could membranes have acted like an enzyme?

Could a membrane act as a catalyst?The issues are:a)How could proteins be held in position by the

membrane?b)How could the frequency of interaction be

increased by that holding?c) What options for distortion of a peptide bond

could be produced by a membrane?These are questions that must wait on

membrane lectures.