Physical Properties - Winthrop University

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Transcript Physical Properties - Winthrop University

Protein Structure: Myoglobin as an Example • John Kendrew solved the structure of myoglobin in 1959 – No Computers!

• Myoglobin is a globular protein consisting of 153 amino acids and a prosthetic group: A Heme • Myoglobin has no  -sheets and is 100%  -helical with respect to secondary structure (we won’t count turns) – There are 8 helices, labelled A through H • Polar amino acids are on the surface and hydrophobic amino acids are in the core of the protein – This is a standard arrangement in proteins • Two histidines help lock the heme group into position • Hydrophobic interactions b/w the heme group and hydrophobic amino acids in the core complete the binding of the heme

The Heme Group

• Heme consists of a metal ion, Fe (II), and a porphyrin ring – The ring is a planar structure • Fe (II) can accommodate 6 coordinate bonds, forming an octahedral arrangement – The porphyrin nitrogens provide 4 of these • An imidazole nitrogen of a histidine in helix F provides a 5th bond • Oxygen binds to Fe (II) to complete the arrangement

Hydrophobic Interactions help anchor the porphyrin ring His 97 and Arg 45 help anchor the ring His 93 Interacts with the Fe (II)

The histidine above the porphyrin ring (on the same side of the ring as where oxygen binds) This off-centered binding forces any other molecule that would bind to the Fe (II) to bind less optimally •Carbon monoxide is a good example •It also allows oxygen to dissociate formt he iron •If the binding was too strong, the Fe (II) - Oxygen bond wouldn’t break

Quick Thoughts on Protein Folding

• There are literally millions of possible ways a simple protein can fold, but only one conformation that works • Hydrophobic interactions help drive protein folding – Not so much the hydrophobic groups attracting each other (That only involves London Forces, right?) – The dipole-dipole interactions between water molecules in solution are much stronger and push the hydrophobic side chains aside • The entropy of the universe must increase in a spontaneous process, and protein folding is a spontaneous process – When water molecules surround a nonpolar compound, they are restricted in the number of hydrogen bonds then can form which represents a lower entropy – By having the hydrophobic residues sequestered in the core of the folded protein, the water molecules are free to form up to 4 hydrogen bonds each.

• This freedom represents greater entropy, thus helping drive folding of the protein

Protein Folding: Myoglobin

QuickTime™ and a YUV420 codec decompressor are needed to see this picture.

QuickTime™ and a YUV420 codec decompressor are needed to see this picture.

Note Placement of hydrophobic residues (green) Where are the side chains pointing?

Note Placement of polar residues (blue) Where are the side chains pointing?

Chapter 6: Enzymes as Catalysts

• Proteins perform many functions in the cell: – Structural roles, Signalling, DNA binding, Energy Transduction, Metabolism and many, many more • Perhaps the most important role proteins play is to serve as catalysts – Enzymes are protein catalysts – Enzymes increase the reaction rate by up to 10 20 times • Non-enzymatic catalysts typically only increase the rate 100 to 10000 fold

Kinetics versus Thermodynamics

Standard Free Energy of Reaction  G ° =  G ° Products  G ° Reactants • The reaction rate depends of the Activation Energy (E A ): E A =  G ° Transition State  G ° Reactants • An enzyme lowers the activation energy – Helps substrate move to transition state

Enzyme Catalyzed Reactions

• An enzyme cannot make a nonspontaneous reaction occur • Let’s look at the reaction of glucose and oxygen: C 6 H 12 O 6 + 6O 2  G ° --> 6CO = -2880 kJ/mole 2 + 6H 2 O • This is a spontaneous reaction, but we have all seen sugar sitting on a tabletop in the open air – The sugar doesn’t spontaneously combust because the Activation energy for the process is too high

Enzyme Catalyzed Reactions

• An enzyme decreases the activation energy barrier • This allows the reaction to proceed at an appreciable rate • By lowering the thermodynamic barrier, we can greatly increase the rate (kinetics) of the reaction

The Effect of Temperature

• Most reaction rates increase as the temperature increases • For nearly every enzyme, this is true up to a point… – Thermal denaturation

Kinetics: Expressing/Describing the Rate of a Reaction

(See the excellent review link on the “Useful Links” page) • A rate is traditionally expressed as the: change in concentration or amount of a substance time • The substance can be a reactant or a product  – If it is a reactant, the rate will have a ____ sign A + B --> P Rate =  [A] t   [B] t   [P] t Rate refers to rate of product formation or rate of reactant disappearance 

Kinetics: Rate Equation

A + B --> P The rate of the reaction is slightly different than strictly looking at the rate of disappearance of reactant or formation of product Rate of Reaction = k[A] f [B] g – where f and g must be empirically determined

Reaction Orders

• The reaction order is an indicator of the details of the reaction mechanism – How many molecules are involved in the reaction – The role of the catalyst in the reaction – Specifics of the system • We’ll only be concerned with 0 th , 1 st 2 nd order reactions.

and

Zero Order Reactions

A --> B Reaction rate = k[A] 0 • The reaction rate is independent of the substrate concentration – Catalyst concentration is what matters in this case • For enzyme catalyzed reactions, we may see such a reaction order when the substrate concentration is VERY high and the enzyme molecules are completely saturated • The cars over the bridge analogy (Six lanes down to 2)

1st Order Reactions

A --> P Reaction rate = k[A] 1 • This reaction is first order with respect to reactant A – What does this mean?

2nd Order Reactions

Glycogen n + P i --> Glucose-1-Phosphate + Glycogen n-1 Reaction rate = k[Glycogen n ] 1 [P i ] 1 • Both glycogen AND Pi have a role in the reaction – The reaction is first order with respect to each reactant, but it is a Second order reaction overall – What does this mean?

• Change either reactant concentration and what would happen to the rate?

Section 6.4: Enzyme-Substrate Binding

• There are 3 major players to consider when evaluating enzyme-catalyzed reactions: 1. The enzyme: The catalyst 2. Substrate: The Reactant / Starting Material 3. Product: The product of the reaction / What is released from the enzyme after the reaction • For the rest of the chapter, we’re going to focus on the interplay between these species

Scheme of an Enzyme-Catalyzed Reaction

1. The Enzyme

BINDS

the substrate, forming the E·S complex (Note the terminology: Big Thing BINDS Little Thing, not the other way around) 2.

The E·S complex forms the Transition State (EX ‡ ) species, which then rapidly forms the product 3. The Product rapidly dissociates from the enzyme, regenerating the catalyst

Formation of the E·S Complex

• The substrate is bound to the active site of the enzyme – Usually (but not always) by covalent means • There are two models that have been created to describe this process:

Formation of the E·S Complex: Lock and Key model

• The active site has a complementary shape to the substrate.

– It exactly fits the substrate – Doesn’t take into account Conformational Flexibility!

– Has fallen into disfavor due to its simplicity

Formation of the E·S Complex: Induced Fit model

• The active site changes shape are the substrate binds, thereby allowing a low energy complex to form.

• This model allows for substrate variability – AD H will react with several aliphatic alcohols – Cytochrome P450s can handle various drugs • There is a limit: Splenda vs Sucrose

Formation of the E·S Complex

• What would happen if the E·S complex was perfect?

– Think about the energy in the diagram – What would E A be?

Formation of the Transition State E

X

and Product Release

• The bond substrate must adopt a conformation of the transition state – By this we mean A LOT of things… • The substrate and the reactive residues of the enzyme are in close proximity • Partial bonds are forming, other bondsare breaking, atoms are shifting around • Proximity and Orientation determine rate • Due to its high energy level, the transition state is just as its name implies: Transitory • As soon as the transition state complex is formed, the product is released

Let’s Look at 2 Different Enzyme Catalyzed Reactions

• • 1. Chymotrypsin Catalyzes the hydrolysis of peptide bonds AND ester bonds – Peptide hydrolysis is its primary function We can take advantage of the ester hydrolysis function to monitor the Activity of the enzyme using p nitrophenylesters • • 2. Aspartate transcarbamoylase The enzyme catalyzes the formation of carbamoyl aspartate from carbamoyl phosphate and aspartate We can monitor the activity of the enzyme directly by spectrophotometry

Chymotrypsin-catalyzed Ester Hydrolysis

• • At low [Substrate], the activity is low As more substrate is added, the rate increases until it reaches a maximum

Aspartate Transcarbamoylase

• • As the [Substrate] increases, the activity does not increase as much until a critical concentration is reached The sigmoidal curve seen for this reaction is indicative of something else going on…

ALLOSTERY!

Allostery

• Allostery is defined as: “ than the enzymatically active one.” Of or involving a change in the shape of and activity of an enzyme that results from molecular binding with a regulatory substance at a site other Huh?

• When a substrate (or an inhibitor) binds to the enzyme somewhere OTHER than the active site, a conformational change may occur that allows more substrate to bind (or less) at other subunits in the quaternary structure and increase (or decrease) the activity.

Positively allosterically regulated by ATP and negatively by CTP •ATP binds to the R subunits and causes the C subunits to open up and bind substrate •CTP causes the C subunits to close up

Allostery

The sigmoidal curve seen for ATCase is an example of Positive Cooperativity caused by allostery