Lecture 6

Protein function and enzyme kinetics

Proteins as enzymes

There are 6 major classes of enzymes:

    1.Oxidoreductases, which are involved in oxidation, reduction, and electron or proton transfer reactions;

   2.Transferases, catalyzing reactions in which groups are transferred;

   3.Hydrolases that cleave various covalent bonds by hydrolysis;

   4.Lyases catalyze reactions forming or breaking double bonds;

   5.Isomerases catalyze isomerization reactions;

   6.Ligases join constituents together covalently.

Enzymes fall into classes based on function

There are 6 major classes of enzymes:

1.Oxidoreductases which are involved in oxidation, reduction, and electron or proton transfer reactions;

   2.Transferases, catalysing reactions in which groups are transferred;

   3.Hydrolases which cleave various covalent bonds by hydrolysis; 4

4.Lyases catalyse reactions forming or breaking double bonds;

5.Isomerases catalyse isomerisation reactions;

   6.Ligases join substituents together covalently.

Enzyme Kinetics

Enzymes are protein catalysts that, like all catalysts, speed up the rate of a chemical reaction without being used up in the process.

Enzyme reaction rates are determined by several factors.

the concentration of substrate molecules (the more of them available, the quicker the enzyme molecules collide and bind with them). The concentration of substrate is designated [S] and is expressed in unit of molarity.

the temperature. As the temperature rises, molecular motion - and hence collisions between enzyme and substrate - speed up. But as enzymes are proteins, there is an upper limit beyond which the enzyme becomes denatured and ineffective.

Enzymes cont.

the presence of inhibitors.

competitive inhibitors are molecules that bind to the same site as the substrate - preventing the substrate from binding as they do so - but are not changed by the enzyme.

noncompetitive inhibitors are molecules that bind to some other site on the enzyme reducing its catalytic power.

pH. The conformation of a protein is influenced by pH and as enzyme activity is crucially dependent on its conformation, its activity is likewise affected.

How we determine how fast an enzyme works

We set up a series of tubes containing graded concentrations of substrate, [S] . At time zero, we add a fixed amount of the enzyme preparation.

Over the next few minutes, we measure the concentration of product formed. If the product absorbs light, we can easily do this in a spectrophotometer.

Early in the run, when the amount of substrate is in substantial excess to the amount of enzyme, the rate we observe is the initial velocity of Vi.

Mechaelis Menton kinetics

Plotting Vi as a function of [S], we find that

At low values of [S], the initial velocity,Vi, rises almost linearly with increasing [S].

But as [S] increases, the gains in Vi level off (forming a rectangular hyperbola).

The asymptote represents the maximum velocity of the reaction, designated Vmax

The substrate concentration that produces a Vi that is one-half of Vmax is designated the Michaelis-Menten constant, Km(named after the scientists who developed the study of enzyme kinetics).

Km is (roughly) an inverse measure of the affinity or strength of binding between the enzyme and its substrate. The lower the Km, the greater the affinity (so the lower the concentration of substrate needed to achieve a given rate).

Plotting out our data it might look like this.

Lineweaver-Burke plot

Competitive inhibitors

Enzymes can be inhibited competitively, when the substrate and inhibitor compete for binding to the same active site or noncompetitively, when the inhibitor binds somewhere else on the enzyme molecule reducing its efficiency.

The distinction can be determined by plotting enzyme activity with and without the inhibitor present.

Competitive Inhibition

In the presence of a competitive inhibitor, it takes a higher substrate concentration to achieve the same velocities that

were reached in its absence. So while Vmax can still be reached if sufficient substrate is available, one-half Vmax requires a higher [S] than before and thus Km is larger.

Non-competitive inhibitor

With noncompetitive inhibition, enzyme molecules that have been bound by the inhibitor are taken out of the game so enzyme rate (velocity) is reduced for all values of [S], including Vmax and one-half Vmax but

Km remains unchanged because the active site of those enzyme molecules that have not been inhibited is unchanged.

Competitive/noncompetitive inhibitor

Effect of inhibitors

How do proteins function?

Structural: Actin is an example it is a major component of the cells architecture as well as the contractile apparatus

Carriers: Hemoglobin is an example. It functions to carry O₂ to tissue and eliminate CO₂

Regulatory: Transcription factors bind to DNA a control the level of mRNA that is produced

Transport: EGFR-epithelial growth factor receptor. Binds EGF and signals for cell growth.

Binders: Immunoglobulin proteins or antibodies- bind to foreign proteins and destroy infectious agents.

Actin and myosin: the contractile apparatus

Skeletal Muscle Cells

Skeletal Muscle Structure

Muscle cells are formed by fusion of myoblasts

Myofibrils are parallel arrays of long cylinders packed in the muscle cell

Sarcomeres are symmetric repeating units from z-line to z-line in the myofibril

Thick filaments are myosin filaments

Thin filaments are actin filaments

Structure of Myosin

More myosin structure

Arrangement of Myosin Molecules in Thick Filaments

bipolar polymer of myosin

myosin tails align and point to center of sarcomere

myosin heads arranged in a helical pattern pointing away from center

myosin heads reach out from the thick filaments to contact the actin filaments

contain ~300 molecules of myosin

Myosin filament

Thin Filaments

actin filaments in the sarcomere are of fixed length

actin filaments are cross-linked by a-actinin at Z-line

both ends of actin filaments are capped

barbed ends are embedded at the Z-line

tropomyosin and troponins bind along each filament

Structure of actin filament

Actin in detail

Actin structure

Folding of the actin molecule is represented by ribbon tracing of the a-carbon atoms. N and C correspond to the amino- and carboxyl-terminals, respectively. The letters followed by numbers represent amino acids in the polypeptide chain. A hypothetical vertical line divides the actin molecule into two domains "large", left side, and "small", right side. ATP and Ca2+ are located between the two domains. These two domains can be subdivided further into two subdomains each, the small domain being composed of subdomains 1 and 2, and the 2 has significantly less mass than the other three subdomains and this is the reason of dividing actin into small and large domains). The four subdomains are held together and stabilized mainly by salt bridges and hydrogen bonds to the phosphate groups of the bound ATP and to its associated Ca2+ localized in the center of the molecule.

Actin domains

1. Where does it polymerize with actin?

2. Where does it interact with troponin and tropomyosin?

3. Where does it interact with myosin?

4. How could we answer this question?

Structure of a Sarcomere

Muscle Contraction

Stabilization of the Alignment of Thick and Thin Filaments

Crystal Structure of Myosin Head and Lever Arm

Regulation of Non-muscle Myosin II Assembly

Muscle continue

Myosin Superfamily

Three examples of the diverse structures of members of the myosin superfamily

In vitro Motility Assay

1. Attach myosin S1 on the cover slip

2. Add fluorescently tagged actin filament

3. Addition of ATP initiates the movement of the filaments

4. Also done by coating cover slip with actin filaments and use fluorescently tagged myosin motor domain


	There are 6 major classes of enzymes:
	1.Oxidoreductases, which are involved in oxidation, reduction, and electron or proton transfer reactions;
	2.Transferases, catalyzing reactions in which groups are transferred;
	3.Hydrolases that cleave various covalent bonds by hydrolysis;
	4.Lyases catalyze reactions forming or breaking double bonds;
	5.Isomerases catalyze isomerization reactions;
	6.Ligases join constituents together covalently.


	There are 6 major classes of enzymes:

	1.Oxidoreductases which are involved in oxidation, reduction, and electron or proton transfer reactions;
	2.Transferases, catalysing reactions in which groups are transferred;
	3.Hydrolases which cleave various covalent bonds by hydrolysis; 4
	4.Lyases catalyse reactions forming or breaking double bonds;
	5.Isomerases catalyse isomerisation reactions;
	6.Ligases join substituents together covalently.


	Enzymes are protein catalysts that, like all catalysts, speed up the rate of a chemical reaction without being used up in the process.


	the concentration of substrate molecules (the more of them available, the quicker the enzyme molecules collide and bind with them). The concentration of substrate is designated [S] and is expressed in unit of molarity.
	the temperature. As the temperature rises, molecular motion - and hence collisions between enzyme and substrate - speed up. But as enzymes are proteins, there is an upper limit beyond which the enzyme becomes denatured and ineffective.


	the presence of inhibitors.
		competitive inhibitors are molecules that bind to the same site as the substrate - preventing the substrate from binding as they do so - but are not changed by the enzyme.
		noncompetitive inhibitors are molecules that bind to some other site on the enzyme reducing its catalytic power.
	pH. The conformation of a protein is influenced by pH and as enzyme activity is crucially dependent on its conformation, its activity is likewise affected.


	We set up a series of tubes containing graded concentrations of substrate, [S] . At time zero, we add a fixed amount of the enzyme preparation.
	Over the next few minutes, we measure the concentration of product formed. If the product absorbs light, we can easily do this in a spectrophotometer.
	Early in the run, when the amount of substrate is in substantial excess to the amount of enzyme, the rate we observe is the initial velocity of Vi.


	Plotting Vi as a function of [S], we find that
	At low values of [S], the initial velocity,Vi, rises almost linearly with increasing [S].
	But as [S] increases, the gains in Vi level off (forming a rectangular hyperbola).
	The asymptote represents the maximum velocity of the reaction, designated Vmax
	The substrate concentration that produces a Vi that is one-half of Vmax is designated the Michaelis-Menten constant, Km(named after the scientists who developed the study of enzyme kinetics).
	Km is (roughly) an inverse measure of the affinity or strength of binding between the enzyme and its substrate. The lower the Km, the greater the affinity (so the lower the concentration of substrate needed to achieve a given rate).


	Enzymes can be inhibited competitively, when the substrate and inhibitor compete for binding to the same active site or noncompetitively, when the inhibitor binds somewhere else on the enzyme molecule reducing its efficiency.
	The distinction can be determined by plotting enzyme activity with and without the inhibitor present.
	Competitive Inhibition
	In the presence of a competitive inhibitor, it takes a higher substrate concentration to achieve the same velocities that
	were reached in its absence. So while Vmax can still be reached if sufficient substrate is available, one-half Vmax requires a higher [S] than before and thus Km is larger.


	With noncompetitive inhibition, enzyme molecules that have been bound by the inhibitor are taken out of the game so enzyme rate (velocity) is reduced for all values of [S], including Vmax and one-half Vmax but
	Km remains unchanged because the active site of those enzyme molecules that have not been inhibited is unchanged.


	Structural: Actin is an example it is a major component of the cells architecture as well as the contractile apparatus
	Carriers: Hemoglobin is an example. It functions to carry O₂ to tissue and eliminate CO₂
	Regulatory: Transcription factors bind to DNA a control the level of mRNA that is produced
	Transport: EGFR-epithelial growth factor receptor. Binds EGF and signals for cell growth.
	Binders: Immunoglobulin proteins or antibodies- bind to foreign proteins and destroy infectious agents.


	Muscle cells are formed by fusion of myoblasts
	Myofibrils are parallel arrays of long cylinders packed in the muscle cell
	Sarcomeres are symmetric repeating units from z-line to z-line in the myofibril
	Thick filaments are myosin filaments
	Thin filaments are actin filaments


	bipolar polymer of myosin
	myosin tails align and point to center of sarcomere
	myosin heads arranged in a helical pattern pointing away from center
	myosin heads reach out from the thick filaments to contact the actin filaments
	contain ~300 molecules of myosin




	actin filaments in the sarcomere are of fixed length
	actin filaments are cross-linked by a-actinin at Z-line
	both ends of actin filaments are capped
	barbed ends are embedded at the Z-line
	tropomyosin and troponins bind along each filament


	1. Where does it polymerize with actin?
	2. Where does it interact with troponin and tropomyosin?
	3. Where does it interact with myosin?
	4. How could we answer this question?



	1. Attach myosin S1 on the cover slip
	2. Add fluorescently tagged actin filament
	3. Addition of ATP initiates the movement of the filaments
	4. Also done by coating cover slip with actin filaments and use fluorescently tagged myosin motor domain